KR20220032640A - Cd3 binding antibodies - Google Patents

Abstract

The present invention relates to novel human CD3 antigen binding polypeptides and preparations thereof and their use in the treatment and/or diagnosis of various diseases, also bispecific antibody molecules capable of activating immune effector cells and diagnosis of various diseases and/or to their use in therapy.

Description

CD3 BINDING ANTIBODIES

This application claims priority to U.S. Provisional Application No. 62/394,360, filed on September 14, 2016, and U.S. Provisional Application No. 62/491,908, filed on April 28, 2017, these applications are The specification is incorporated by reference in its entirety.

The body's immune system acts as a defense against infection, damage, and cancer. Two distinct but interrelated systems, the humoral and cellular immune systems, work together to protect the body. The humoral system is mediated by soluble factors called antibodies, which neutralize products recognized by the body as foreign. In contrast, cellular systems require cells, such as T cells and macrophages, to clear and neutralize foreign invaders.

Activation of T cells is crucial for the stimulation of immune responses. T cells exhibit immunological specificity and are the driving force behind most of the cellular immune response. Although T cells do not secrete antibodies, they are required for secretion of antibodies by B lymphocytes. T cell activation requires the participation of a number of cell surface molecules, such as the T cell receptor complex and CD4 or CD8 molecules. Antigen-specific T cell receptors (TcRs) are composed of disulfide-linked heterodimeric, membrane glycoproteins with chains, alpha and beta (α and β) or gamma and delta (γ and δ). TcR is non-covalently linked with a complex of constant proteins designated as CD3.

T cells are known to exert potent antitumor effects in a variety of experimental settings. Antibodies capable of effectively recruiting T cells against tumor cells include, for example, tumor-associated antigens (TAA) and agonistic T-cell membrane proteins such as the TCR/CD3 complex and bispecific antibodies directed against CD28. was available as These bispecific antibodies can activate T cells irrespective of their TCR specificity, resulting in specific lysis of cells bearing individual TAAs.

However, although anti-CD3 bispecific antibodies can redirect T-cell-mediated lysis towards malignant cells, clinical trials with CD3-based bsAbs have shown high toxicity in patients. Non-specific T-cell activation from bsAbs can occur in an antigen-independent manner due to Fc/Fc receptor (FcR) interactions, or in an antigen-dependent manner when the antigen is expressed on both normal and tumor cells. Both mechanisms may have been responsible for the toxicity observed in previous clinical studies. (See, eg, Link et al. (1998) Int. J. Cancer 77(2):251-6; Durben et al. Molecular Therapy (2015); 23 4, 648-655). Because of the resulting cytokine release syndrome, there have been significant obstacles to the development of these antibodies for therapeutic purposes.

The interaction of the T cell receptor (TCR) with its peptide-MHC ligand determines the activity of the T cell. The binding nature of this interaction has been studied in more detail and has been shown to control T cell function. The strength and nature of the TCR-peptide/MHC interaction determines whether T cells exert effector functions or are inactivated and deleted. Antibodies to CD3 activate T cells by changing the conformation of the CD3ε chain and, depending on the epitope, may have either agonistic or antagonistic effects on T cells (Yoon et al., 1994 Immunity 1:563-569). ). In view of the significant side effects of many T cell agonists, it may be desirable to reduce the release of proinflammatory cytokines while maintaining potent antitumor effects. However, partial agonist anti-CD3 antibodies alter the CD3ε chain, resulting in suboptimally ineffective signaling, and most anti-CD3 antibodies are full agonists for both pathways. It is unclear whether these effector functions can be separated. Many existing anti-CD3 antibodies (eg, SP-34, UCHT1, OKT3) have affinities in the range of 1-50 nM KD, however, they may not be optimal for therapeutic use.

CD3 specific antibodies, and bispecific antibodies derived therefrom, are provided by the present invention.

publication

CD3 antibodies are disclosed, for example, in U.S. Patent Nos. 5,585,097; 5,929,212; 5,968,509; 6,706,265; 6,750,325; 7,381,803; 7,728,114. Bispecific antibodies with CD3 binding specificity are disclosed, eg, in US Pat. Nos. 7,262,276; 7,635,472; 7,862,813; and 8,236,308, each of which is specifically incorporated herein by reference.

Embodiments of the present invention provide compositions for a family of closely related antibodies that bind to and activate signaling through CD3, eg, activate CD3 ⁺ T cells, and methods of using the same.

An antibody family comprises a set of CDR sequences as defined herein. The family of antibodies provides a number of benefits that contribute to utility clinically as therapeutic agent(s). Antibodies within this family include members with varying binding affinities, allowing the selection of specific sequences with the desired affinity. The ability to fine-tune the affinity is particularly important for managing the level of CD3 activation in the subject being treated, thereby reducing toxicity.

In some embodiments, the anti-CD3 antibody has an affinity (KD) for CD3 in the range of about 10 ^-6 to about 10 ^-11 M. Anti-CD3 antibodies with affinity (KD) of 50 nM or more, 100 nM or more, 500 nM or more, or 1 μM or more more closely mimic TCR/MHC interaction, and effective tumor cell It may be desirable to minimize toxic cytokine release while maintaining dissolution. In some embodiments, the anti-CD3 antibody is characterized or selected for a reduced propensity to induce cytokine release upon binding to competent T cells, eg, release of IL-2 and IFNγ. Antibodies that optimize the killing of tumor cells and reduced release of cytokines, e.g., within a family of antibody sequences described herein, less than nearly half the maximum observed for family members in relative assays, and relative assays Antibodies that induce cytokine release that may be less than the maximum observed for a family member in a , eg, less than the maximum and about 25% may be selected for therapeutic use. In some embodiments, bispecific or multispecific antibodies are provided, which comprise at least a heavy chain variable region from an antibody family, and may comprise a heavy and light chain variable region provided herein. A bispecific antibody comprises at least a heavy chain variable region of an antibody specific for a protein other than CD3, and may comprise a heavy chain and a light chain variable region. In some such embodiments, the second antibody specifically binds a tumor associated antigen, a targeting antigen, such as an integrin, a pathogen antigen, a checkpoint protein, and the like. Various types of bispecific antibodies are within the scope of the present invention, including without limitation single chain polypeptides, two chain polypeptides, three chain polypeptides, four chain polypeptides, and combinations thereof.

Each CD3-specific antibody comprises a VH domain comprising CDR1, CDR2 and CDR3 sequences in a human VH framework. Family 2 CDR sequences include, for example, amino acid residues 26 to 33 of the provided exemplary variable region sequences, set forth in SEQ ID NOs: 1-18 for CDR1, CDR2 and CDR3, respectively; 51 to 58; and 97 to 112 periphery. It will be understood by those skilled in the art that these CDR sequences may be in different positions if different framework sequences are selected, although in general the order of the sequences will remain the same.

A CDR sequence for a family 2 antibody may have the formula X represents a variable amino acid, which may be a specific amino acid as shown below.

CDR1

G ₁ F ₂ T ₃ F ₄ X ₅ X ₆ Y ₇ A ₈

during the meal,

X ₅ can be any amino acid; In some embodiments, X ₅ is D, A, or H; In some embodiments X ₅ is D.

X ₆ can be any amino acid; In some embodiments X ₆ is D or N; In some embodiments D ₆ is D.

In some embodiments, the CDR1 sequence of a family 2 anti-CD3 antibody comprises the sequence set forth in any of SEQ ID NOs: 1-18, residues 26-33.

CDR2

I _1' S _2' W _3' N _4' S _5' G _6' S _7' I _8'

In some embodiments, the CDR2 sequence of the family 2 anti-CD3 antibody comprises a sequence set forth in any of SEQ ID NOs: 1-18, residues 51-58.

CDR3

A _1" K _2" D _3" S _4" R _5" G _6" Y _7" G _8" X _9" Y _10" X _11" X _12" G _13" G _12" A _15" Y _16"

during the meal,

X _9″ can be any amino acid, in some embodiments, X _9″ is D or S; In some embodiments, X _9″ is D;

X _11″ can be any amino acid, in some embodiments X _11″ is R or S;

X _12″ can be any amino acid, and in some embodiments X _12″ is L or R.

In some embodiments, the CD3 sequence of the family 2 anti-CD3 antibody has the formula AKDSRGYGDYX _11" X _12" GGAY, wherein X _11" and X _12" are as defined above. In some embodiments, the CDR3 sequence of the family 2 anti-CD3 antibody comprises the sequence set forth in any of SEQ ID NOs: 1-18, residues 97-112. In some embodiments, the CD3-binding VH domain of a family 2 antibody is paired with a light chain variable region domain. In some such embodiments, the light chain is a fixed light chain.

In some embodiments the light chain comprises a VL domain having CDR1, CDR2 and CDR3 sequences in a human VL framework. The CDR sequence may have SEQ ID NO: 19. In some embodiments, the CDR1 sequence comprises amino acid residues 27-32 for CDR1, CDR2, and CDR3, respectively; 50-52; 89-97.

In some embodiments the CDR sequence of an antibody of the invention is a sequence having at least 85% identity, at least 90% identity, at least 95% identity, at least 99% identity to a CDR sequence or set of CDR sequences in SEQ ID NOs: 1-18 . In some embodiments a CDR sequence of the present invention comprises 1, 2, 3 or more amino acid substitutions relative to a CDR sequence or set of CDR sequences in any one of SEQ ID NOs: 1-18. In some embodiments the amino acid substitution(s) is one or more of positions 5 or 10 of CDR1, positions 2, 6 or 7 of CDR2, positions 1, 8, 9 or 10 of CDR3, relative to the Family 2 formulas provided above .

In some embodiments, a bispecific antibody of the invention comprises a CD3-binding variable region as described herein, paired with a light chain. In some embodiments the light chain comprises a variable region sequence set forth in SEQ ID NO: 19, or a variable region comprising a set of CDR sequences and a framework sequence in SEQ ID NO: 19. A variety of Fc sequences find use in, without limitation, human IgG1, IgG2a, IgG2b, IgG3, IgG4, and the like. In some embodiments, the second arm of the bispecific antibody comprises a variable region that specifically binds a tumor-associated antigen. In some embodiments, the second arm of the bispecific antibody comprises a variable region that specifically binds BCMA. In some embodiments the anti-BCMA arm is a single chain variable region, eg, as depicted in FIG. 2B . In some embodiments the anti-BCMA arm comprises a variable region sequence set forth in SEQ ID NO: 20; or the tandem variable region sequence set forth in SEQ ID NO:21. The Fc sequence of the anti-BCMA arm can be, without limitation, human IgG1, IgG2a, IgG2b, IgG3, IgG4, and the like. The CDR sequences may be those nested in SEQ ID NO: 20. In some embodiments, the CDR sequence comprises amino acid residues 26-33 for each of CDR1, CDR2, CDR3; 51-58; 97-108.

In another embodiment, a monospecific, bispecific, etc. antibody or antibody-like protein comprising at least a CD3-binding VH domain of the invention, eg, at least a CD3-binding VH domain of the invention; And there is provided a pharmaceutical composition comprising a pharmaceutically acceptable excipient. Compositions may be lyophilized, suspended in solution, etc., and may be presented in unit dose formulations.

In some embodiments, a method is provided for the treatment of cancer, the method comprising administering to a subject in need thereof an effective dose of a monospecific, bispecific, etc. antibody of the invention. Where the antibody is bispecific, the second antigen binding site may specifically bind a tumor antigen, checkpoint protein, or the like. In various embodiments, the cancer is ovarian cancer, breast cancer, gastrointestinal cancer, brain cancer, head and neck cancer, prostate cancer, colon cancer, lung cancer, leukemia, lymphoma, sarcoma, carcinoma, neuronal cell tumor, squamous cell carcinoma, germ cell tumor, metastasis, and an undifferentiated tumor, a seminoma, a melanoma, a myeloma, a neuroblastoma, a mixed cell tumor, and a neoplasm caused by an infectious agent.

In some embodiments, a method is provided for the treatment of an infectious disease, the method comprising administering to a subject in need thereof an effective dose of a monospecific, bispecific, etc. antibody of the invention. Where the antibody is bispecific, the second antigen binding site may specifically bind a pathogen antigen, such as a bacterium, virus or parasite.

In another embodiment, there is provided a method for the production of a bispecific antibody of the present invention, wherein the method comprises generating an antibody sequence, e.g., one or more light chain encoding sequences, one or more heavy chain encoding sequences, in a single host cell. including expression. In various embodiments, the host cell may be a prokaryotic or eukaryotic cell, eg, a mammalian cell.

CD3 binding antibodies according to embodiments of the present invention may provide a number of benefits that contribute to their utility as therapeutic agent(s).

BRIEF DESCRIPTION OF THE DRAWINGS The present invention is best understood from the following detailed description when read in conjunction with the accompanying drawings. A patent or application file contains at least one drawing made in color. Copies of these patents or patent application publications with colored drawing(s) will be provided by the Secretariat upon request and payment of fees. In accordance with common practice, various features of these figures are emphasized not to scale. In contrast, the dimensions of the various features are arbitrarily enlarged or reduced for clarity. These drawings include the following drawings.
1a to 1c . 1A shows residues 26-33; 51-58; and alignment of the CDR1, 2 and 3 regions of antibody family 2 members of SEQ ID NOs: 1-18 that specifically bind human CD3 corresponding to 97-112. 1B shows CDR1, 2 and 3 regions of an immobilized light chain (SEQ ID NO: 19); and exemplary anti-BCMA sequences (SEQ ID NO: 20 and SEQ ID NO: 21). 1C provides the CDR sequence (SEQ ID NO: 22), ID 304704, of a reference anti-CD3 antibody.
2a to 2e . A diagram depicting a phylogenetic model of a bispecific human antibody. Figure 2A Anti-CD3:anti-tumor-antigen bispecific antibody with conventional light chains (3 unique chains in total). Figure 2B Anti-CD3:anti-tumor-antigen bispecific antibody with two distinct light chains (four unique chains in total). Figure 2C Anti-CD3:anti-tumor-antigen bispecific antibody with heavy chain alone tumor antigen binding domain chain (3 distinct chains). Figure 2D Anti-CD3:anti-tumor-antigen bispecific antibody with scFv tumor antigen binding domains (3 unique chains in total). Figure 2E Anti-CD3:anti-tumor-antigen bispecific antibody with scFv anti-CD3 binding domain (3 unique chains in total).
3 . Anti-CD3 Family 2 data table summarizing the behavior of anti-CD3 antibodies in monospecific and bispecific formats. Column 1 shows the sequence ID for the anti-CD3 VH sequence. Column 2 shows the MFI values for binding to Jurkat cells of the parental monospecific anti-CD3. Column 3 shows the MFI values for cyno T-cell binding of parental monospecific anti-CD3. Column 4 shows the names of the aCD3:aBCMA bispecific antibodies. Column 5 shows a picogram of IL-2 released by pan T-cells stimulated by a bispecific antibody binding to BCMA protein coated on plastic at the indicated doses. Column 6 shows a picogram of IL-6 released by pan T-cells stimulated by a bispecific antibody binding to BCMA protein coated on plastic at the indicated doses. Column 7 shows a picogram of IL-10 released by pan T-cells stimulated by a bispecific antibody binding to BCMA protein coated on plastic at the indicated doses. Column 8 shows a picogram of IFN-γ released by pan T-cells stimulated by a bispecific antibody binding to BCMA protein coated on plastic at the indicated doses. Column 9 shows a picogram of TNFα released by pan T-cells stimulated by a bispecific antibody binding to BCMA protein coated on plastic at the indicated doses. Column 10 shows the EC50 of bispecific antibody-mediated U266 tumor cell lysis in the presence of human pan T-cells. Column 11 shows the percent lysis of U266 tumor cells in the presence of the bispecific antibody and human pan T-cells at a dose of 333 ng/mL of the bispecific antibody. Column 12 shows the protein binding affinity of the anti-CD3 arm of the bispecific antibody as measured by Octet. Column 13 shows the MFI values for binding to Jurkat cells of the bispecific antibody.
Figure 4. Bispecific antibody-mediated tumor cell lysis. Seven αCD3_fam2:aBCMA bispecific antibodies, each with a unique anti-CD3 arm and a conventional anti-BCMA arm, were tested for their ability to kill U266 BCMA+ tumor cells through redirection of activated primary T cells. In these experiments U266 cells expressing BCMA were mixed with activated pan T-cells at a 10:1 E:T ratio with the addition of bispecific antibody. The x-axis shows the concentration of antibody used, and the y-axis shows the lysis % of tumor cells 6 hours after addition of the antibody.
Figure 5 . Bispecific U266 killing activity correlated with IL-2 release. Comparison of bispecific antibody-mediated tumor cell lytic activity with IL-2 cytokine release is shown in the scatter plot. The correlation between IL-2 production and U266 tumor cell lysis is R ² =0.37.
Figure 6. Bispecific U266 killing activity correlated with IFN-release. Comparison of bispecific antibody-mediated tumor cell lytic activity with IFN-γ cytokine release is shown in scatter plots. The correlation between IFN-g production and U266 tumor cell lysis is R ² =0.53.
Figure 7. Bispecific U266 killing activity correlated with anti-CD3 binding affinity. Comparison of bispecific antibody-mediated U266 tumor cell lytic activity with anti-CD3 binding affinity is shown in the scatter plot. The correlation between U266 killing EC50 and protein binding affinity is R ² =0.93.
8a to 8d. Bispecific antibody-mediated tumor cell lysis. The αCD3_F1F:aBCMA bispecific antibody was assayed for its ability to kill three different BCMA+ tumor cells and one BCMA-negative cell line through redirection of activated primary T cells. In these experiments, tumor cells were mixed with activated pan T-cells at a 10:1 E:T ratio with the addition of bispecific antibody. Figure 8a shows the death of RPMI-8226 cells, Figure 8b shows the death of NCI-H929 cells, Figure 8c shows the death of U-266 cells, and Figure 8d shows the death of the negative control K562 cells shows The x-axis shows the concentration of antibody used, and the y-axis shows the lysis % of tumor cells 6 hours after addition of the antibody.
9a to 9d. Bispecific antibody-mediated IL-2 release. Levels of IL-2 cytokine release were measured after resting human T cells were incubated with various tumor cell lines and increasing doses of the αCD3_F1F:aBCMA bispecific antibody. FIG. 9A shows IL-2 release stimulated by RPMI-8226 cells, FIG. 9B shows IL-2 release stimulated by NCI-H929 cells, and FIG. 9C shows IL stimulated by U-266 cells. -2 release, and FIG. 9D shows IL-2 release stimulated by the negative control, K562 cells.
10a to 10d. Bispecific antibody-mediated IFN-γ release. Levels of IFN-γ cytokine release were measured after resting human T cells were incubated with various tumor cell lines and increasing doses of the αCD3_F1F:aBCMA bispecific antibody. 10A shows IFN-γ release stimulated by RPMI-8226 cells, FIG. 10B shows IFN-γ release stimulated by NCI-H929 cells, and FIG. 10C shows IFN-γ release stimulated by U-266 cells. -γ release, and FIG. 10D shows IFN-γ release stimulated by the negative control, K562 cells.

To facilitate understanding of the present invention, a number of terms are defined below.

Before the active agents and methods of the present invention are described, it is to be understood that the invention is not limited to the particular methods, products, devices and factors described, as such methods, devices, and formulations may, of course, vary. . It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the scope of the present invention, which will be limited only by the appended claims.

As used in this specification and the appended claims, it should be noted that the singular forms include plural referents unless the context dictates otherwise. Thus, for example, reference to "a drug candidate" refers to one or a mixture of such candidates, and reference to "method" includes instructions to equivalent steps and methods known to those skilled in the art, and so forth.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. All publications mentioned herein are incorporated herein by reference for the purpose of describing and disclosing devices, formulations, and methods that are described in such publications and that may be used in connection with the invention described herein.

Where a range of values is provided, unless the context dictates otherwise, the value lies between each of up to tenths of a unit of the lower limit between the upper and lower limits of that range, and any other stated or Intervening values are understood to be encompassed within the scope of the present invention. The upper and lower limits of these smaller ranges may independently be included within the smaller ranges, and are also subject to any specifically excluded limit within the stated range and encompassed within the scope of the invention. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the invention.

In the following description, various specific details are set forth in order to provide a more thorough understanding of the present invention. However, it will be apparent to one skilled in the art that the present invention may be practiced without one or more of these specific details. In other instances, features and procedures well known to those skilled in the art have not been described in order to avoid obscuring the present invention.

In general, conventional methods of protein synthesis, recombinant cell culture and protein isolation, and recombinant DNA technology within the skill in the art are used in the present invention. Such techniques are fully described in the existing literature, eg, Maniatis, Fritsch & Sambrook, Molecular Cloning: A Laboratory Manual (1982); Sambrook, Russell and Sambrook, Molecular Cloning: A Laboratory Manual (2001); Harlow, Lane and Harlow, Using Antibodies: A Laboratory Manual: Portable Protocol No. I, Cold Spring Harbor Laboratory (1998); and Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory; (1988)].

Justice

"Comprising" means that the recited elements are required in the composition/method/kit, but other elements may be included within the scope of the claims to form the composition/method/kit and the like.

"Consisting essentially of" is meant to limit the scope of a described composition or method to the specified materials or steps that do not materially affect the basic and novel feature(s) of the invention.

"Consisting of" is meant to exclude from a composition, method, or kit any element, step, or component not specified in a claim.

The terms "treatment", "treating" and the like are used herein to mean obtaining a desired pharmacological and/or physiological effect in general. The effect may be prophylactic in terms of completely or partially preventing a disease or symptom thereof and/or therapeutic in terms of partial or complete cure for the disease and/or adverse effects resulting from the disease. As used herein, “treatment” encompasses any treatment of a disease in a mammal, and (a) preventing the disease from developing in an individual predisposed to the disease but not yet diagnosed as having it; (b) inhibiting the disease, ie, stopping its development; or (c) alleviating the disease, ie, causing remission of the disease. Therapeutic agents may be administered before, during or after the onset of the disease or injury. Of particular interest is the treatment of progressive disease in which the treatment stabilizes or reduces the unwanted clinical symptoms of a patient. Such treatment is preferably performed prior to complete loss of function in the affected tissue. The subject therapy may be administered during the symptomatic stage of the disease and in some cases after the symptomatic stage of the disease.

A “therapeutically effective amount” is intended to be that amount of an active agent necessary to confer a therapeutic benefit to a subject. For example, a “therapeutically effective amount” is an amount that induces, ameliorates, or otherwise causes improvement in pathological symptoms, disease progression, or physiological condition associated with a disease, or improves resistance to a disorder.

The terms “subject,” “individual,” and “patient” are used interchangeably herein to refer to a mammal being assessed and/or treated for treatment. In one embodiment, the mammal is a human. The terms “subject,” “individual,” and “patient” encompass, without limitation, individuals suffering from cancer, individuals suffering from autoimmune diseases, individuals suffering from pathogenic infections, and the like. The subject may be a human, but also includes other mammals, particularly mammals useful as laboratory models for human diseases, such as mice, rats, and the like.

The terms “cancer,” “neoplasm,” and “tumor” are used herein to refer to cells that exhibit autonomous, unregulated growth, such as to exhibit a deviant growth phenotype characterized by a significant loss of control over cell proliferation. used interchangeably. Cells of interest for detection, analysis or treatment herein include precancerous (eg, benign), malignant, premetastatic, metastatic and non-metastatic cells. Cancers of virtually all tissues are known. The phrase “cancer burden” refers to the quantum or cancer volume of cancer cells in an individual. Reducing the cancer burden thus refers to reducing the number or cancer volume of cancer cells in an individual. As used herein, the term “cancer cell” refers to any cell that is or is derived from a cancer cell, eg, a clone of a cancer cell. Many types, including solid tumors such as carcinomas, sarcomas, glioblastomas, melanomas, lymphomas, myelomas, and the like, and leukemias, including circulating cancers such as specifically B cell leukemia, T cell leukemia, and the like. of cancers are known to those skilled in the art. Examples of cancer include ovarian cancer, breast cancer, colon cancer, lung cancer, prostate cancer, hepatocellular carcinoma, stomach cancer, pancreatic cancer, cervical cancer, ovarian cancer, liver cancer, bladder cancer, cancer of the urinary tract, thyroid cancer, kidney cancer, carcinoma, melanoma, head and neck cancer and brain cancer.

“Antibody-dependent cell-mediated cytotoxicity” and “ADCC” refer to non-specific cytotoxic cells expressing Fc receptors, such as natural killer cells, neutrophils and macrophages, which recognize bound antibodies on target cells and lyse the target cells. refers to a cell-mediated response that elicits ADCC activity can be assessed using methods such as those described in US Pat. No. 5,821,337. ADCP refers to antibody dependent cell-mediated phagocytosis.

An “effector cell” is a leukocyte that expresses one or more constant region receptors and performs an effector function.

A “cytokine” is a protein that is released by one cell and acts as an intercellular mediator in another cell. Cytokines of interest include, but are not limited to, cytokines released from activated T cells, eg, IL-2, IFNγ, and the like.

"Non-immunogenic" refers to a substance that does not initiate, elicit or enhance an immune response, wherein the immune response includes an adaptive and/or innate immune response.

The term “isolated” means that the substance has been transferred from its original environment (eg, its natural environment, if it occurs naturally). For example, a naturally occurring polynucleotide or polypeptide present in a living animal is not isolated, but the same polynucleotide or polypeptide isolated from some or all of the coexisting material in a natural system is isolated. Such polynucleotides may be part of a vector and/or such polynucleotides or polypeptides may be part of a composition and still be isolated in that such vector or composition is not part of its natural environment.

"Pharmaceutically acceptable excipient" means an excipient that is generally safe, non-toxic, and useful in preparing the desired pharmaceutical composition, and includes excipients acceptable for veterinary as well as human pharmaceutical use. do. Such excipients may be solid, liquid, semi-solid, or, in the case of aerosol compositions, a gas.

"Pharmaceutically acceptable salts and esters" means salts and esters that are pharmaceutically acceptable and have the desired pharmacological properties. Such salts include salts that may be formed when an acidic proton present in the compound is capable of reacting with an inorganic or organic base. Suitable inorganic salts include those formed with alkali metals such as sodium and potassium, magnesium, calcium, and aluminum. Suitable organic salts include those formed with organic bases such as amine bases such as ethanolamine, diethanolamine, triethanolamine, tromethamine, N methylglucamine, and the like. Such salts also include inorganic acids (such as hydrochloric acid and hydrobromic acid) and organic acids (such as acetic, citric, maleic, and alkane- and arene-sulfonic acids, such as methanesulfonic acid and benzenesulfonic acid). acid addition salts formed with Pharmaceutically acceptable esters include esters formed from the carboxy, sulfonyloxy and phosphonooxy groups present in the compound, for example, a C _1-6 alkyl ester. When two acidic groups are present, the pharmaceutically acceptable salt or ester may be a mono-acid-mono-salt or ester or di-salt or ester; And similarly, when two or more acidic groups are present, some or all of these groups may be chlorinated or esterified. The compounds named in the present invention may exist in unchlorinated or unesterified forms, or in chlorinated and/or esterified forms, and the nomenclature of such compounds is inherently (unchlorinated and unesterified) compounds and their pharmaceutically acceptable forms. It is intended to include both salts and esters. In addition, certain compounds named herein may exist in one or more stereoisomeric forms, and the naming of such compounds includes all single stereoisomers and all mixtures of such stereoisomers, whether racemic or otherwise. it is intended to be

The terms "pharmaceutically acceptable", "physiologically tolerable" and grammatical variations thereof are used interchangeably when they refer to compositions, carriers, diluents and reagents, and which substances would prevent administration of the composition. to the extent that it can be administered to humans without the occurrence of undesirable physiological effects.

"Homology" between two sequences is determined by sequence identity. If the two sequences compared to each other differ in length, the sequence identity preferably relates to the percentage of nucleotide residues of the shorter sequence that are identical to the nucleotide residues of the longer sequence. Sequence identity can traditionally be determined using a computer program, such as the Bestfit program (Wisconsin Sequence Analysis Package, version 8 for Unix, Genetics Computer Group, University Research Park, 575 Science Drive Madison, Wis. 53711). Bestfit utilizes the local homology algorithm of Smith and Waterman, Advances in Applied Mathematics 2 (1981), 482-489 to find the segment with the highest sequence identity between two sequences. When using Bestfit or other sequence alignment programs to determine whether a particular sequence has, for example, 95% identity to a reference sequence of the invention, the parameter is preferably that the percentage of identity is calculated over the full length of the reference sequence, and Adjustments are made to allow for a homology gap of up to 5% of the total number of nucleotides in the reference sequence. When using Bestfit, the so-called optional parameters preferably remain at their preset (“default”) values. Deviations appearing in a comparison between a given sequence and the aforementioned sequences of the invention may be caused by, for example, additions, deletions, substitutions, insertions or recombination. Such sequence comparisons are preferably performed in the program "fasta20u66" (version 2.0u66 by William R. Pearson and University of Virginia, September 1998; WR Pearson (1990), Methods in Enzymology 183, 63-98, the attached examples and See also http://workbench.sdsc.edu/) can also be implemented. For this purpose, a “default” parameter setting may be used.

A “variant” refers to a polypeptide having an amino acid sequence that differs somewhat from a native sequence polypeptide. Ordinarily, amino acid sequence variants will possess at least about 80% sequence identity, more preferably, at least about 90% homology by sequence. Amino acid sequence variants may possess substitutions, deletions and/or insertions at certain positions within the reference amino acid sequence.

As used herein, the term “vector” is intended to refer to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked. One type of vector is a “plasmid,” which refers to a circular double-stranded DNA loop into which additional DNA segments can be ligated. Another type of vector is a viral vector, in which additional DNA segments can be ligated into the viral genome. Certain vectors (eg, bacterial vectors with bacterial origins of replication and episomal mammalian vectors) are capable of autonomous replication in the host cell into which they are introduced. Other vectors (eg, non-episomal mammalian vectors) can be integrated into the genome of a host cell upon introduction into the host cell, and thus are replicated along with the host genome. In addition, certain vectors are capable of directing the expression of genes to which they are operatively linked. Such vectors are referred to herein as "recombinant expression vectors" (or simply, "recombinant vectors"). In general, expression vectors of utility in recombinant DNA technology are often in the form of plasmids. In this specification, "plasmid" and "vector" may be used interchangeably, since plasmid is the most commonly used form of vector.

As used herein, the term “host cell” (or “recombinant host cell”) refers to a cell that has been, or can be genetically altered, by introduction of an exogenous polynucleotide, eg, a recombinant plasmid or vector. It is intended to refer to It should be understood that this term is intended to refer to a particular subject cell as well as the progeny of such cell. Because certain modifications may occur in subsequent generations due to mutations or environmental influences, such progeny may not in fact be identical to the parent cell, but are still included within the scope of the term "host cell" as used herein.

"Binding affinity" generally refers to the strength of the sum total of non-covalent interactions between a single binding site of a molecule (eg, an antibody or other binding molecule) and its binding partner (eg, an antigen or receptor). The affinity of a molecule X for its relative Y can generally be expressed by the dissociation constant (Kd). Affinity can be measured by conventional methods known in the art, including those described herein. Low affinity antibodies bind antigen (or receptor) weakly and tend to dissociate easily, whereas high affinity antibodies bind antigen (or receptor) more tightly and remain bound longer.

Unless specifically indicated to the contrary, the term "conjugate" as described and claimed herein is defined as a heterogeneous molecule formed by the covalent attachment of one or more antibody fragment(s) to one or more polymer molecule(s). wherein the heterogeneous molecule is water soluble, ie is soluble in a physiological fluid, such as blood, and wherein the heterogeneous molecule is free of any structured aggregates. A conjugate of interest is PEG. In the context of the preceding definition, the term "structured aggregate" refers to (1) an aqueous solution having a spheroid or spheroid shell structure such that heterogeneous molecules are not within micelles or other emulsion structures and are not anchored to lipid bilayers, vesicles or liposomes. any aggregate of molecules in; and (2) solid or insolubilized form that does not release the heterologous molecule into solution upon contact with the aqueous phase, eg, any aggregate of molecules in a chromatography bead matrix. Thus, as defined herein, the term "conjugate" encompasses the aforementioned heterologous molecules in a precipitate, sediment, bioerodible matrix or other solid capable of releasing the heterologous molecule into an aqueous solution upon hydration of the solid.

The word “label,” as used herein, refers to a detectable compound or composition that is conjugated directly or indirectly to an antibody. The label may be detectable by itself (eg, a radioisotope label or a fluorescent label) or, in the case of an enzymatic label, may catalyze a chemical alteration of a substrate compound or composition that is detectable.

"Solid phase" is meant to be a non-aqueous matrix to which an antibody of the invention can adhere. Examples of solid phases encompassed herein include those formed partially or completely from glass (eg, controlled porosity glass), polysaccharides (eg, agarose), polyacrylamide, polystyrene, polyvinyl alcohol, and silicone. In certain embodiments, depending on the context, the solid phase may comprise the wells of an assay plate; In others it is a purification column (eg, an affinity chromatography column). This term also includes discrete solid phases of discrete particles, such as those described in US Pat. No. 4,275,149.

Antibodies, also referred to as immunoglobulins, traditionally comprise at least one heavy chain and one light chain, wherein the amino-terminal domains of the heavy and light chains are variable in sequence and thus variable region domains, or variable heavy (VH) chains or variable Commonly referred to as the light chain (VH) domain. Although, as discussed herein, specific binding can be achieved with the heavy chain alone variable sequence, and although a variety of non-natural conformations of antibodies are known and used in the art, these two domains have traditionally been associated with specific form a bonding region.

"Functional" or "biologically active" antibodies or antigen binding molecules (including heavy chain single antibodies and bispecific three-chain antibody-like molecules (TCAs) herein) are those that occur naturally in a structural, regulatory, biochemical or biophysical event. It is capable of exerting one or more of the activities. For example, a functional antibody or other binding molecule, eg, TCA, may have the ability to specifically bind an antigen, and such binding, in turn, elicits a cellular or molecular event, eg, signal transduction or enzymatic activity. may or may not be changed. A functional antibody or other binding molecule, such as a TCA, may also block ligand activation of a receptor, or act as an agonist or antagonist. The ability of an antibody or other binding molecule, eg, TCA, to exert one or more of its natural activities depends on several factors, including proper folding and assembly of the polypeptide chains.

The term "antibody" is used herein in its broadest sense, and is used in monoclonal antibodies, polyclonal antibodies, monomers, dimers, multimers, multispecific antibodies (eg, bispecific antibodies), heavy chain monoclonal antibodies, three chain antibodies , single chain Fvs, Nanobodies, etc., and also include antibody fragments as long as they exhibit the desired biological activity (Miller et al (2003) Jour. of Immunology 170:4854-4861). The antibody may be murine, human, humanized, chimeric, or an antibody derived from another species.

The term antibody includes a full-length heavy chain, a full-length light chain, an intact immunoglobulin molecule; or an immunologically active portion of any of these polypeptides, i.e., a polypeptide comprising an antigen binding site that immunospecifically binds to an antigen or portion thereof of a target of interest, such target being autoimmune cancer cells or cells that produce autoimmune antibodies associated with the disease, but are not limited thereto. The immunoglobulins disclosed herein can be of any type (eg, IgG, IgE, IgM, IgD and IgA), class (eg, IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2), or reduced or enhanced It is possible to have a subclass of immunoglobulin molecules, including an engineered subclass with an altered Fc region that provides effector cell activity. Immunoglobulins may be derived from any species. In one aspect, the immunoglobulin is primarily of human origin.

The term “variable” refers to the fact that certain portions of the variable domains differ widely in sequence between antibodies and are utilized for the binding and specificity of each particular antibody for a particular antigen. However, the variability is not evenly distributed throughout the variable domains of antibodies. It is concentrated in three segments called hypervariable regions in both the light and heavy chain variable domains. The more highly conserved portions of variable domains are called framework regions (FR). The variable domains of the native heavy and light chains each comprise four FRs that predominantly adopt a beta-sheet configuration, joined by three hypervariable regions, which form loop linkages, and in some cases, a beta-sheet structure. form part of The hypervariable regions in each chain are bound in close proximity by the FRs, and together with the hypervariable regions from the other chain, contribute to the formation of the antigen binding site of the antibody (Kabat et al (1991) Sequences of Proteins of Immunological). Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md]. The constant domains are not directly involved in binding the antibody to antigen, but exhibit various effector functions, such as participation of the antibody in antibody dependent cellular cytotoxicity (ADCC).

As used herein, the term “hypervariable region” refers to the amino acid residues of an antibody that are responsible for antigen binding. A hypervariable region may comprise amino acid residues from a “complementarity determining region” or “CDR” and/or residues from a “hypervariable loop”. "Framework region" or "FR" residues are variable domain residues other than hypervariable region residues as defined herein.

A variable region of interest comprises at least one CDR sequence, typically at least two CDR sequences, and more typically three CDR sequences from a Family 2 variable region provided herein. Although exemplary CDR designations are shown herein, one of ordinary skill in the art will appreciate that various definitions of CDRs are commonly used, including the Kabat definition, which is based on sequence variability and is the most commonly used ("Zhao et al. A germline knowledge based computational approach for determining antibody complementarity determining regions." See Mol Immunol. 2010;47:694-700). The Chothia definition is based on the location of structural loop regions (Chothia et al. "Conformations of immunoglobulin hypervariable regions." Nature. 1989;342:877-883). Alternative CDR definitions of interest are described, without limitation, by Hoegger, "Yet another numbering scheme for immunoglobulin variable domains: an automatic modeling and analysis tool." J Mol Biol. 2001;309:657-670; Ofran et al. "Automated identification of complementarity determining regions (CDRs) reveals peculiar characteristics of CDRs and B cell epitopes." J Immunol. 2008;181:6230-6235; Almagro "Identification of differences in the specificity-determining residues of antibodies that recognize antigens of different size: implications for the rational design of antibody repertoires." J Mol Recognit. 2004;17:132-143; and Padlan et al. "Identification of specificity-determining residues in antibodies." Faseb J. 1995;9:133-139, each of which is specifically incorporated herein by reference.

As used herein, the term "monoclonal antibody" refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies constituting the population are free from possible naturally occurring mutations that may be present in small amounts. same except Monoclonal antibodies are highly specific and directed against a single antigenic site. Moreover, in contrast to polyclonal antibody preparations comprising different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen. Monoclonal antibodies are advantageous in that, in addition to their specificity, they can be synthesized uncontaminated by other antibodies. The modifier "monoclonal" indicates the character of the antibody as being obtained from a substantially homogeneous population of the antibody, and is not to be construed as requiring production of the antibody by any particular method.

An antibody herein refers to an antibody in which portions of its heavy and/or light chains are identical to or homologous to the corresponding sequences in antibodies derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain(s) is A "chimeric" antibody that is identical to or homologous to a corresponding sequence in an antibody from another species or belonging to another antibody class or subclass, as well as a fragment of such antibody, can be specifically engineered to exhibit the desired biological activity. (U.S. Pat. No. 4,816,567; and Morrison et al (1984) Proc. Natl. Acad. Sci. USA, 81:6851-6855). Chimeric antibodies of interest herein include "primatized" antibodies comprising variable domain antigen binding sequences derived from a non-human primate (eg, macaque, apes, etc.) and human constant region sequences.

As used herein, an “intact antibody chain” is one comprising a full-length variable region and a full-length constant region (Fc). Intact "traditional" antibodies comprise an intact light chain and an intact heavy chain to secreted IgG, as well as a light chain constant domain (CL) and heavy chain constant domains, CH1, Hinge, CH2 and CH3. Different isotypes, such as IgM or IgA, may have different CH domains. The constant domain may be a native sequence constant domain (eg, a human native sequence constant domain) or an amino acid sequence variant thereof. An intact antibody may have one or more "effector functions", which refer to biological activities attributable to the Fc constant region (native sequence Fc region or amino acid sequence variant Fc region) of the antibody. Examples of antibody effector functions include: Clq binding; complement dependent cytotoxicity; Fc receptor binding; antibody dependent cell mediated cytotoxicity (ADCC); phagocytosis; and downregulation of cell surface receptors. Constant region variants include those that alter effector profile, binding to Fc receptors, and the like.

Depending on the amino acid sequence of the Fc (constant domain) of the heavy chain, antibodies and various antigen-binding proteins can be presented as different classes. There are five major classes of heavy chain Fc regions: IgA, IgD, IgE, IgG and IgM, and some of these "subclasses" (isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA and IgA2. can be further subdivided into The Fc constant domains corresponding to the different classes of antibodies are called α, δ, ε, γ and μ, respectively. The structures and three-dimensional shapes of these subunits of the different classes of immunoglobulins are well known. Ig conformations include hinge-modified or hingeless conformations (Roux et al (1998) J. Immunol. 161:4083-4090; Lund et al (2000) Eur. J. Biochem. 267:7246-7256; US 2005 /0048572; US 2004/0229310). The light chains of antibodies from any vertebrate species can be assigned to one of two types, called κ and λ, based on the amino acid sequence of their constant domains.

A “functional Fc region” possesses the “effector function” of a native sequence Fc region. Exemplary effector functions include C1q binding; CDC; Fc-receptor binding; ADCC; ADCP; downregulation of cell surface receptors (eg, B-cell receptors); and the like. Such effector functions generally depend on whether the Fc region is a receptor, eg, Fcγ RI; FcγRIIA; FcγRIIB1; FcγRIIB2; FcγRIIIA; Required to interact with the Fcγ RIIIB receptor, and the low affinity FcRn receptor; and can be assessed using various assays, eg, as disclosed in the definitions herein. A “dead” Fc is one that is mutated to retain activity, eg, for extending serum half-life, but does not activate the high affinity Fc receptor.

A “native sequence Fc region” comprises an amino acid sequence identical to the amino acid sequence of an Fc region found in nature. Native-sequence human Fc regions include, for example, native-sequence human IgG1 Fc regions (non-A and A allotypes); native-sequence human IgG2 Fc region; native-sequence human IgG3 Fc region; and native-sequence human IgG4 Fc regions as well as naturally occurring variants thereof.

A “variant Fc region” comprises an amino acid sequence that differs from that of the native sequence Fc region by at least one amino acid modification, preferably one or more amino acid substitution(s). Preferably, the variant Fc region comprises at least one amino acid substitution in the native sequence Fc region or in the Fc region of the parent polypeptide compared to the native sequence Fc region or the Fc region of the parent polypeptide, e.g., from about 1 to about 10 amino acid substitutions, and preferably about 1 to about 5 amino acid substitutions. The variant Fc region herein preferably has at least about 80% homology with the native sequence Fc region and/or with the Fc region of the parent polypeptide, and most preferably at least about 90% homology thereto, more preferably with it will possess at least about 95% homology with

The variant Fc sequence may comprise three amino acid substitutions in the CH2 region to reduce Fcγ RI binding at EU index positions 234, 235 and 237 (see Duncan et al., (1988) Nature 332:563). Two amino acid substitutions at EU index positions 330 and 331 within the complement Clq binding site reduce complement fixation (Tao et al., J. Exp. Med. 178:661 (1993) and Canfield and Morrison, J. Exp. Med. 173:1483 (1991)). Substitution of an IgG2 residue at positions 233-236 and an IgG4 residue at positions 327, 330 and 331 into human IgG1 significantly reduces ADCC and CDC (see, e.g., Armor KL. et al ., 1999 Eur J Immunol. 29(8)). :2613-24; and Shields RL et al ., 2001. J Biol Chem. 276(9):6591-604). Other Fc variants are possible, including, without limitation, deletions of regions capable of forming disulfide bonds, or deletion of certain amino acid residues at the N-terminal end of the native Fc form or addition of methionine residues thereto. Accordingly, in one embodiment of the present invention, one or more Fc portion of the scFc molecule may comprise one or more mutations in the hinge region to eliminate disulfide bonds. In another embodiment, the hinge region of the Fc may be completely removed. In another embodiment, the molecule may comprise an Fc variant.

Moreover, Fc variants can be constructed such that effector function is eliminated or substantially reduced by substituting, deleting or adding amino acid residues that result in complement binding or Fc receptor binding. For example, and without limitation, deletions may occur at complement-binding sites, eg, C1q-binding sites. Techniques for preparing such sequence derivatives of immunoglobulin Fc fragments are disclosed in International Patent Publication Nos. WO 97/34631 and WO 96/32478. In addition, the Fc domain may be modified by phosphorylation, sulfation, acylation, glycosylation, methylation, farnesylation, acetylation, amidation, and the like.

Fc may be in a form having native sugar chains, increased sugar chains compared to the native form, or decreased sugar chains compared to the native form, or may be in an aglycosylated or deglycosylated form. The increase, decrease, removal or other modification of sugar chains can be achieved by a method conventional in the art, for example, a chemical method, an enzymatic method, or by expressing it in a genetically engineered production cell line. Such cell lines may include microorganisms that naturally express glycosylation enzymes, such as Pichia Pastoris, and mammalian cell lines, such as CHO cells. In addition, the microorganism or cell can be engineered to express a glycosylation enzyme, or made incapable of expressing a glycosylation enzyme (see, e.g., Hamilton, et al., Science, 313:1441 (2006); Kanda, et al, J. Biotechnology, 130:300 (2007); Kitagawa, et al., J. Biol. Chem., 269 (27): 17872 (1994); Ujita-Lee et al., J. Biol. Chem. , 264(23): 13848 (1989); Imai-Nishiya, et al, BMC Biotechnology 7:84 (2007); and WO 07/055916). As one example of cells engineered to have altered sialylation activity, the alpha-2,6-sialyltransferase 1 gene was engineered into Chinese hamster ovary cells and into sf9 cells. Antibodies expressed by these engineered cells are thus sialylated by the exogenous gene product. A further method for obtaining an Fc molecule having an altered amount of sugar residues compared to a plurality of native molecules is to separate the plurality of molecules into glycosylated and aglycosylated fractions using, for example, lectin affinity chromatography. (see, eg, WO 07/117505). The presence of certain glycosylation moieties has been shown to alter the function of immunoglobulins. For example, removal of sugar chains from an Fc molecule results in a sharp decrease in the binding affinity for the Clq portion of the first complement component C1 and a decrease or loss in antibody-dependent cell-mediated cytotoxicity (ADCC) or complement-dependent cytotoxicity (CDC). and thus do not induce an unnecessary immune response in vivo. Further important modifications include sialylation and fucosylation: the presence of sialic acid in IgG correlated with anti-inflammatory activity (see, eg, Kaneko, et al, Science 313:760 (2006)), whereas fucosylation from IgG Ablation of the course results in enhanced ADCC activity (see, eg, Shoj-Hosaka, et al, J. Biochem., 140:777 (2006)).

In an alternative embodiment, the antibodies of the invention may have Fc sequences with enhanced effector function, eg, by increasing their binding capacity to Fcγ RIIIA and increasing ADCC activity. For example, fucose attached to the N -linked glycan at Asn-297 of Fc sterically interferes with the interaction of Fc with FcγRIIIA, and removal of fucose by glycoprocessing can increase binding to FcγRIIIA, This translates to >50-fold high ADCC activity compared to wild-type IgG1 control. Protein processing through amino acid mutations in the Fc portion of IgG1 yielded multiple variants that increase the affinity of Fc binding to Fcγ RIIIA. In particular, the triple alanine mutant S298A/E333A/K334A exhibits a 2-fold increased binding to Fcγ RIIIA and ADCC function. The S239D/I332E (2X) and S239D/I332E/A330L (3X) variants have a significant increase in binding affinity to Fcγ RIIIA and enhancement of ADCC ability in vitro and in vivo. Other Fc variants identified by yeast display also showed enhanced binding to Fcγ RIIIA and enhanced tumor cell death in a mouse xenograft model. See, eg, Liu et al. (2014) JBC 289(6):3571-90.

The term “Fc-region-comprising antibody” refers to an antibody comprising an Fc region. The C-terminal lysine of the Fc region (residue 447 according to the EU numbering system) can be removed, for example, during purification of the antibody or by recombinantly engineering the nucleic acid encoding the antibody. Accordingly, an antibody having an Fc region according to the present invention may include an antibody with or without K447.

An “Fv” is the smallest antibody fragment containing a complete antigen-recognition and antigen-binding site. The CD3 binding antibody of the present invention comprises a dimer of one heavy chain and one light chain variable domain in tight, non-covalent association; However, by way of example, additional antibodies for use in a multispecific configuration may comprise a VH in the absence of a VL sequence. Even a single variable domain (or half of an Fv comprising only three hypervariable regions specific for an antigen) has the ability to recognize and bind antigen, although it may have lower affinity than that of the two domain binding sites.

The Fab fragment also contains the constant domain of the light chain and the first constant domain of the heavy chain (CH1). Fab' fragments differ from Fab fragments by the addition of a few residues at the carboxy terminus of the heavy chain CH1 domain comprising one or more cysteines from the antibody hinge region. Fab'-SH is herein the designation for Fab' in which the cysteine residue(s) of the constant domain bear at least one free thiol group. F(ab') ₂ antibody fragments were initially produced as pairs of Fab' fragments, which have hinge cysteines between them. Other chemical linkages of antibody fragments are also known.

"Humanized" forms of non-human (eg, rodent) antibodies, including single chain antibodies, are chimeric antibodies (including single chain antibodies) that contain minimal sequence derived from non-human immunoglobulin. See, eg, Jones et al, (1986) Nature 321:522-525; Chothia et al (1989) Nature 342:877; Riechmann et al (1992) J. Mol. Biol. 224, 487-499; Foote and Winter, (1992) J. Mol. Biol. 224:487-499; Presta et al (1993) J. Immunol. 151, 2623-2632; Werther et al (1996) J. Immunol. Methods 157:4986-4995; and Presta et al (2001) Thromb. Haemost. 85:379-389. For further details, see US Pat. Nos. 5,225,539; 6,548,640; 6,982,321; 5,585,089; 5,693,761; 6,407,213; Jones et al (1986) Nature, 321:522-525; and Riechmann et al (1988) Nature 332:323-329.

As used herein, the term “single chain antibody” refers to a single polypeptide chain containing one or more antigen binding domains that bind to an epitope of an antigen, wherein such domains are derived from the variable region of an antibody heavy or light chain. derived or have sequence identity with these regions. The portion of this variable region is V _H or V _L gene segments, D and J _H gene segment, or J _L It can be encoded by a gene segment. The variable region is a rearranged V _H DJ _H , V _L DJ _H , V _H J _L , or V _L J _L It can be encoded by a gene segment. V-, D- and J-gene segments can be derived from humans and a variety of animals including birds, fish, sharks, mammals, rodents, non-human primates, camels, llamas, rabbits, and the like.

The CD3-binding antibodies of the invention find particular utility in multispecific configurations including, without limitation, bispecific antibodies, trifunctional antibodies, and the like. A wide variety of methods and protein shapes are known and used in bispecific monoclonal antibodies (BsMAB), trispecific antibodies, and the like.

The first-generation BsMAbs consisted of two heavy and two light chains, one each from two different antibodies. These two Fab regions are directed against two antigens. The Fc region is constructed from two heavy chains and forms a third binding site with an Fc receptor on immune cells (see, e.g., Lindhofer et al., The Journal of Immunology, Vol 155, p 219-225, 1995). see). Antibodies may be from the same or different species. For example, cell lines expressing murine and mouse antibodies secrete functional bispecific Abs due to preferential species-limited heavy and light chain matching. In other embodiments the Fc region is designed to fit only in a specific manner.

Another type of bispecific antibody comprises a chemically linked Fab that consists only of a Fab region. Two chemically linked Fab or Fab2 fragments form artificial antibodies that bind to two different antigens and are one type of bispecific antibody. Antigen-binding fragments (Fab or Fab2) of two different monoclonal antibodies are produced and linked by chemical means such as thioethers (Glennie, MJ et al., Journal of immunology 139, p 2367-75, 1987; Peter Borchmann). et al., Blood, Vol. 100, No. 9, p 3101-3107, 2002).

Various other methods for the production of multivalent artificial antibodies have been developed by recombinantly fusing the variable domains of two antibodies. A single chain variable fragment (scFv) is a fusion protein of the variable regions of the heavy (VH) and light (VL) chains of an immunoglobulin, linked by a short linker peptide of 10 to about 25 amino acids. Linkers are typically rich in glycine for flexibility as well as serine or threonine for solubility, and can link the N-terminus of the VH to the C-terminus of the VL and vice versa. Bispecific single chain variable fragments (di-scFv, non-scFv) can be engineered by linking two scFvs with different specificities. A single peptide chain with two VH and two VL regions is produced, yielding a bivalent scFv.

The bispecific tandem scFv is also known as the bispecific T-cell engager (BiTE). A bispecific scFv can be created with a linker peptide in which the two variable regions are too short (about 5 amino acids) to fold together, which forces the scFv to dimerize. This type is known as a diabody (Adams et al., British journal of cancer 77, p 1405-12, 1998). Dual-affinity retargeting (DART) platform technology (Macrogenics, Rockville, Md.). This fusion protein technology utilizes two single chain variable fragments (scFvs) of different antibodies on a single peptide chain of about 55 kilodaltons. SCORPION Therapeutics (Emergent Biosolutions, Inc., Seattle, Wash.) combines two antigen binding domains in a single chain protein. Based on the immunoglobulin Fc region, one binding domain is on the C terminus and a second binding domain is on the N terminus of the effector domain.

Trivalent and bispecific antibody-like proteins also include DVD-Igs processed from two monoclonal antibodies (Wu, C. et al., Nature Biotechnology, 25, p 1290-1297, 2007). To construct a DVD-Ig molecule, the V domains of two mAbs are tandemly fused at the N-terminus with the variable domain of the first antibody heavy chain (VL), followed by the other antibodies VL and Ck, by a short linker (TVAAP). to form a DVD-Ig protein light chain. Similarly, the variable regions of the heavy chains (VH) of the two mAbs are fused in tandem with the first antibody, then other antibodies and heavy chain constant domains at the N-terminus by a short linker (ASTKGP) to the DVD-Ig protein heavy chain ( VH1/VL1). All light and heavy chain constant domains are conserved in the DVD-Ig design, as they are critical for the formation of disulfide-linked, intact IgG-like molecules. Cotransfection of mammalian cells with expression vectors encoding DVD-Ig light and heavy chains results in secretion of a single species of an IgG-like molecule with a molecular weight of nearly 200 kDa. These molecules here have four binding sites, two from each mAb.

The term “bispecific three-chain antibody-like molecule” or “TCA” is used herein to refer to an antibody-like molecule comprising, consisting essentially of, or consisting of three polypeptide subunits. , two of these subunits comprise, or consist essentially of, one heavy chain and one light chain of a monoclonal antibody, or a functional antigen-binding fragment of such an antibody chain comprising an antigen binding region and at least one CH domain consists of, or consists of. This heavy/light chain pair has a binding specificity for the first antigen. wherein the third polypeptide subunit comprises an Fc portion comprising CH2 and/or CH3 and/or CH4 domains in the absence of the CH1 domain, and an antigen binding domain that binds to an epitope of a second antigen or a different epitope of the first antigen It comprises, consists essentially of, or consists of a heavy chain alone antibody, wherein such binding domain is derived from or has sequence identity with the variable region of an antibody heavy or light chain. The portion of this variable region is V _H and/or V _L gene segments, D and J _H gene segment, or J _L It can be encoded by a gene segment. The variable region is a rearranged V _H DJ _H , V _L DJ _H , V _H J _L , or V _L J _L It can be encoded by a gene segment.

The TCA protein utilizes a heavy chain sole antibody" or "heavy chain antibody" or "heavy chain polypeptide", which, as used herein, comprises a single chain comprising heavy chain constant regions CH2 and/or CH3 and/or CH4 without a CH1 domain. means antibody.In one embodiment, heavy chain antibody consists of antigen binding domain, at least part of hinge region, and CH2 and CH3 domain.In another embodiment, heavy chain antibody comprises antigen binding domain, at least part of hinge region, And it consists of CH2 domain.In a further embodiment, the heavy chain antibody consists of antigen binding domain, at least part of hinge region and CH3 domain.The heavy chain antibody of CH2 and/or CH3 domain is truncated is also included herein. In an embodiment, the heavy chain is without hinge region, and consists of antigen binding domain and at least one CH (CH1, CH2, CH3 or CH4) domain.Heavy chain single antibody can be in the form of dimer, wherein two heavy chains are disulfide bond or otherwise covalently or non-covalently attached to each other.Heavy chain antibody may belong to the IgG subclass, but antibodies belonging to other subclasses, such as IgM, IgA, IgD and IgE subclasses are also used herein. In a specific embodiment, the heavy chain antibody is an IgG1, IgG2, IgG3 or IgG4 subtype, in particular an IgG1 subtype.

Heavy chain antibodies constitute about a quarter of the IgG antibodies produced by camelids, such as camels and llamas (Hamers-Casterman C., et al. Nature. 363, 446-448 (1993)). These antibodies are formed by two heavy chains and lack a light chain. As a result, the variable antigen binding moiety is referred to as the VHH domain, and it represents the smallest naturally occurring, intact, antigen binding site of only approximately 120 amino acids in length (Desmyter, A., et al. J. Biol. Chem. 276, 26285-26290 (2001)). Heavy chain antibodies with high specificity and affinity can be generated against various antigens through immunization (van der Linden, RH, et al. Biochim. Biophys. Acta. 1431, 37-46 (1999)), and the VHH portion can be readily cloned and expressed in yeast (Frenken, LGJ, et al. J. Biotechnol. 78, 11-21 (2000)). Their expression levels, solubility and stability are much higher than those of classical F(ab) or Fv fragments (Ghahroudi, M. A. et al. FEBS Lett. 414, 521-526 (1997)). Sharks have also been found to have a single VH-like domain in their antibody, designated VNAR. (Nuttall et al. Eur. J. Biochem. 270, 3543-3554 (2003); Nuttall et al. Function and Bioinformatics 55, 187-197 (2004); Dooley et al., Molecular Immunology 40, 25-33 (2003) )).

Antibodies or antigen binding molecules, including heavy chain single antibodies and bispecific three-chain antibody-like molecules (TCAs) herein, which "bind" to an antigen of interest, are diagnostic and diagnostic in the ability of such antibodies or binding molecules to target antigen. It is useful as a therapeutic agent and/or binds to an antigen with sufficient affinity to not significantly cross-react with other proteins. In such embodiments, the extent of binding of the antibody or other binding molecule to the non-targeted antigen will be within 10% as determined by fluorescence activated cell sorting (FACS) analysis or radioimmunoprecipitation (RIA).

protein

The present invention provides a family of closely related antibodies that bind to CD3 and activate signaling through it, eg, activate CD3 ⁺ T cells. Antibodies within this family comprise a set of CDR sequences as defined herein and are exemplified by the provided VH sequences in SEQ ID NOs: 1-18. The family of antibodies provides a number of benefits that contribute to utility clinically as therapeutic agent(s). Antibodies within this family include members with varying binding affinities, allowing the selection of specific sequences with the desired affinity. The ability to fine-tune the affinity is particularly important for managing the level of CD3 activation in the subject being treated, and thus reducing toxicity. For example, if low abundance tumor antigens (less than 10,000 molecules per cell) are targeted, high affinity CD3 binding agents (<30 nM) would be expected to be desirable. If high abundance tumor antigens (more than 50,000 molecules per cell) are targeted, CD3 binding agents with low affinity (>50 nM) are preferred. The propensity of an antibody to induce the release of cytokines, e.g., IL-2, IFNγ, etc., when bound to T cells, can be assessed independently of affinity, where reduced cytokine release may be desirable. there is.

Suitable antibodies may be selected from those provided herein for development and use, including without limitation, for use as bispecific antibodies. Determination of affinity for a candidate protein may be performed using a method known in the art, for example, Biacore assay or the like. Members of the antibody family include, without limitation, from about 10 ^-6 to about 10 ^-10 ; about 10 ^-6 to about 10 ^-9 ; about 10 ^-6 to about 10 ^-8 ; about 10 ^-8 to about 10 ^-11 ; about 10 ^-8 to about 10 ^-10 ; about 10 ^-8 to about 10 ^-9 ; about 10 ^-9 to about 10 ^-11 ; about 10 ^-9 to about 10 ^-10 ; or have an affinity for CD3 with a Kd of from about 10 ^-6 to about 10 ^-11 including any value within these ranges. Affinity selection can be confirmed, for example, in the assessment of potential toxicity and biological assessment of T cell activation in vitro or in preclinical models. Determination of cytokine release can be assessed using any convenient method including, but not limited to, the assays described in the Examples.

Incorporation of the T cell receptor (TCR) by binding to the MH-peptide complex or anti-TCR/CD3 antibody initiates T cell activation. Examples of anti-TCR/CD3 antibodies that activate T cells are OKT3 and UCHT1. These anti-CD3 antibodies cross-compete for binding to CD3 on T cells and are routinely used in T cell activation assays. The anti-CD3 antibodies of the invention cross-compete with OKT3 for binding to human CD3. Depending on the binding affinity for epitopes on CD3 and CD3, anti-CD3 antibodies activated T cells with different functional outcomes. In vitro culture of human T cells with low affinity anti-CD3 antibody resulted in incomplete activation of T cells, low IL-2 and IL-10 production. In contrast, high affinity CD3 binding agents activated T cells to produce much more IL-2 and other cytokines. Low affinity anti-CD3 antibodies are considered to be partial agonists which selectively induce some effector functions, potent tumor killing and CD69 upregulation, but fail to induce others, e.g., IL-2 and IL-10 production. do. The high affinity binding agents of the present invention are full agonists that activate many immune effector functions of T cells. The intensity of the interaction with CD3 and the recognized epitope resulted in qualitatively different activation of T cells. The maximal cytokine production of T cells activated by the low affinity anti-CD3 antibody was lower than the maximal activation by the high affinity anti-CD3 antibody. In some embodiments, an antibody of the invention elicits lower release of either or both of IL-2 and IL-10 when combined with T cells in an activation assay as compared to a reference anti-CD3 antibody in the same assay. Here, the reference antibody may be ID 304703 (SEQ ID NO: 22) or an antibody of equivalent affinity. the maximal release of IL-2 and/or IL-10 is less than about 75% of the release by the reference antibody, less than about 50% of the release by the reference antibody, less than about 25% of the release by the reference antibody, and It may be less than about 10% of the release by the reference antibody.

In some embodiments of the invention, bispecific or multispecific antibodies are provided, which may have any of the shapes discussed herein, including without limitation three chain bispecifics. A bispecific antibody comprises at least a heavy chain variable region of an antibody specific for a protein other than CD3, and may comprise a heavy chain and a light chain variable region. In some such embodiments, the second antibody specificity binds to a tumor associated antigen, a targeting antigen, eg, an integrin, etc., a pathogen antigen, a checkpoint protein, etc. Various types of bispecific antibodies are within the scope of the present invention, including without limitation single chain polypeptides, two chain polypeptides, three chain polypeptides, four chain polypeptides, and combinations thereof.

The family of CD3-specific antibodies comprises a VH domain comprising CDR1, CDR2 and CDR3 sequences in a human VH framework. The CDR sequences include, by way of example, amino acid residues 26-33 of the provided exemplary variable region sequences set forth in SEQ ID NOs: 1-18, for each of CDR1, CDR2 and CDR3; 51-58; and 97-112 perimeter. It will be understood by those skilled in the art that these CDR sequences may be in different positions if different framework sequences are selected, although in general the order of the sequences will remain the same.

A CDR sequence for a family 2 antibody may have the formula X indicates a variable amino acid, which may be a specific amino acid as indicated below.

CDR1

G ₁ F ₂ T ₃ F ₄ X ₅ X ₆ Y ₇ A ₈

during the meal,

X ₅ can be any amino acid; In some embodiments, X ₅ is D, A, or H; In some embodiments X ₅ is D.

X ₆ can be any amino acid; In some embodiments X ₆ is D or N; In some embodiments D ₆ is D.

In some embodiments, the CDR1 sequence of a family 2 anti-CD3 antibody comprises the sequence set forth in any of SEQ ID NOs: 1-18, residues 26-33.

CDR2

I _1' S _2' W _3' N _4' S _5' G _6' S _7' I _8'

In some embodiments, the CDR2 sequence of the family 2 anti-CD3 antibody comprises a sequence set forth in any of SEQ ID NOs: 1-18, residues 51-58.

CDR3

A _1" K _2" D _3" S _4" R _5" G _6" Y _7" G _8" X _9" Y _10" X _11" X _12" G _13" G _12" A _15" Y _16"

during the meal,

X _9″ can be any amino acid, in some embodiments, X _9″ is D or S; In some embodiments, X _9″ is D;

X _11″ can be any amino acid, in some embodiments X _11″ is R or S;

X _12″ can be any amino acid, and in some embodiments X _12″ is L or R.

In some embodiments, the CD3 sequence of the family 2 anti-CD3 antibody has the formula AKDSRGYGDYX _11" X _12" GGAY, wherein X _11" and X _12" are as defined above. In some embodiments, the CDR3 sequence of the family 2 anti-CD3 antibody comprises the sequence set forth in any of SEQ ID NOs: 1-18, residues 97-112.

In some embodiments the CD3-binding VH domain is opposed to a light chain variable region domain. In some such embodiments the light chain is a fixed light chain. In some embodiments the light chain comprises a VL domain having CDR1, CDR2 and CDR3 sequences in a human VL framework. The CDR sequences may be those of SEQ ID NO: 19. In some embodiments, the CDR1 sequence comprises amino acid residues 27-32 for each of CDR1, CDR2, CDR3; 50-52; 89-97.

In some embodiments the CDR sequence of the family 2 antibody has at least 85% identity, at least 90% identity, at least 95% identity, at least 99% identity to a CDR sequence or set of CDR sequences in one of SEQ ID NOs: 1-18 have a sequence In some embodiments, a CDR sequence of the invention comprises 1, 2, 3 or more amino acid substitutions for a CDR sequence or set of CDR sequences in one of SEQ ID NOs: 1-18. In some embodiments the amino acid substitution(s) is one or more of positions 5 or 10 of CDR1, positions 2, 6 or 7 of CDR2, positions 1, 8, 9 or 10 of CDR3, relative to the sequence provided above .

When the protein of the invention is a bispecific antibody, one binding moiety, ie the VH/VL combination or VH alone, is specific for human CD3, while the other arm is a cancer cell, e.g., ovarian, breast , gastrointestinal tract, brain, head and neck, prostate, colon, lung cancer, etc. as well as hematological tumors such as B-cell tumors including leukemia, lymphomas, sarcomas, carcinomas, neuronal cell tumors, squamous cell carcinomas, reproductive Cell tumors, metastases, undifferentiated tumors, seminomas, melanomas, myelomas, neuroblastomas, mixed cell tumors, neoplasms caused by infectious agents, and other malignancies, cells infected with pathogens, inflammation and/or autoimmunity can be specific for target cells, including autoreactive cells that induce Non-CD3 moieties may also be specific for immune regulatory proteins, as described herein.

Tumor-associated antigens (TAA) are relatively confined to tumor cells, whereas tumor specific antigens (TSA) are unique to tumor cells. TSA and TAA are typically part of intracellular molecules expressed on the cell surface as part of the major histocompatibility complex.

Tissue specific differentiation antigens are molecules present on tumor cells and their normal cellular counterparts. Tumor-associated antigens known to be recognized by therapeutic mAbs fall into several different categories. Hematopoietic differentiation antigens are glycoproteins commonly associated with differentiation cluster (CD) clustering and include CD20, CD30, CD33 and CD52. Cell surface differentiation antigens are a diverse group of glycoproteins and carbohydrates found on the surface of both normal and tumor cells. Antigens involved in growth and differentiation signaling are often growth factors and growth factor receptors. Target growth factors for antibodies in cancer patients are CEA, epidermal growth factor receptor (EGFR; also known as ERBB1), ERBB2 (also known as HER2), ERBB3, MET (also known as HGFR), insulin-like growth factor 1 receptor (IGF1R), ephrin receptor A3 (EPHA3), tumor necrosis factor (TNF)-related apoptosis-inducing ligand receptor 1 (TRAILR1; also known as TNFRSF10A), TRAILR2 (also known as TNFRSF10B) and nucleus receptor activators of factor-κB ligands (RANKL; also known as TNFSF11). Antigens involved in angiogenesis are usually proteins or growth factors that support the formation of new microvasculature, including vascular endothelial growth factor (VEGF), VEGF receptor (VEGFR), integrin αVβ3 and integrin α5β1. Tumor stroma and extracellular matrix are essential support structures for tumors. Interstitial and extracellular matrix antigens that are therapeutic targets include fibroblast activation protein (FAP) and tenascin.

Examples of therapeutic antibodies useful in a bispecific configuration include, without limitation, rituximab; ibritumomab; tiuxetane; tositumomab; brentuximab; vedotin; gemtuzumab; ozogamicin; alemtuzumab; IGN101; adekatumumab; rabetuzumab; huA33; femtumomab; oregobomab; CC49 (minretumomab); cG250; J591; MOv18; MORAb-003 (parletuzumab); 3F8, ch14.18; KW-2871; hu3S193; IgN311; bevacizumab; IM-2C6; CDP791; etaracizumab; voloximab; cetuximab, panitumumab, nimotuzumab; 806; trastuzumab; Pertuzumab; MM-121; AMG 102, METMAB; SCH 900105; AVE1642, IMC-A12, MK-0646, R1507; CP 751871; KB004; IIIA4; mapatumumab (HGS-ETR1); HGS-ETR2; CS-1008; denosumab; cibrotuzumab; F19; and 81C6.

The most actively studied immune-checkpoint receptors in the context of clinical cancer immunotherapy, cytotoxic T-lymphocyte-associated antigen 4 (CTLA4; also known as CD152) and programmed cell death protein 1 (PD1; also known as CD279). present) - are both inhibitory receptors. The clinical activity of antibodies blocking either of these receptors suggests that anti-tumor immunity can be enhanced at multiple levels, and that combinatorial strategies can be intelligently designed and informed by mechanistic considerations and preclinical models. .

The two ligands for PD1 are PD1 ligand 1 (PDL1; also known as B7-H1 and CD274) and PDL2 (also known as B7-DC and CD273). PDL1 is expressed on cancer cells, and through binding to the receptor PD1 on T cells, it inhibits T cell activation/function.

Lymphocyte activation gene 3 (LAG3; also known as CD223), 2B4 (also known as CD244), B and T lymphocyte attenuator (BTLA; also known as CD272), T cell membrane protein 3 (TIM3; also known as HAVcr2) known), adenosine A2a receptor (A2aR) and a family of killer inhibitory receptors, respectively, are associated with inhibition of lymphocyte activity and, in some cases, induction of lymphocyte anergy. Antibody targeting of these receptors can be used in the methods of the invention.

Agents that agonize immune costimulatory molecules are also useful in the methods of the invention. Such agents include agonists or CD40 and OX40. CD40 is a costimulatory protein found in antigen presenting cells (APCs) and is required for their activation. These APCs include phagocytes (macrophages and dendritic cells) and B cells. CD40 is part of the TNF receptor family. The primary activating signaling molecules for CD40 are IFNγ and CD40 ligand (CD40L). Stimulation through CD40 activates macrophages.

Anti-CCR4 (CD194) antibodies of interest include humanized monoclonal antibodies directed towards C-C chemokine receptor 4 (CCR4), with potential anti-inflammatory and anti-neoplastic activity. CCR2 is expressed in inflammatory macrophages that can be found in a variety of inflammatory conditions, such as rheumatoid arthritis; And it was also confirmed to be expressed in tumor-promoting macrophages. CCR2 is also expressed on regulatory T cells, and the CCR2 ligand, CCL2, mediates recruitment of regulatory T cells into tumors. Regulatory T cells inhibit the response to anti-tumor T cells and, therefore, their inhibition or depletion is desired.

Protein production of the present invention

Although antibodies can be prepared by chemical synthesis, they are typically produced by methods of recombinant DNA technology, such as co-expression of all chains that make up a protein in a single recombinant host cell, or heavy chain polypeptides and antibodies, such as For example, it is produced by co-expression of human antibodies. In addition, antibody heavy and light chains can also be expressed using a single polycistronic expression vector. Purification of individual polypeptides is accomplished using standard protein purification techniques such as affinity (protein A) chromatography, size exclusion chromatography and/or hydrophobic interaction chromatography. Bispecifics differ sufficiently in size and hydrophobicity that purification can be performed using standard procedures.

The amount of antibody and heavy chain polypeptide produced in a single host cell can be minimized through processing of the constant regions of the antibody and heavy chain such that homodimerization is favored over heterodimerization, e.g., by introducing self-complementary interactions (e.g., , see WO 98/50431 for possibilities, for example the "protuberance-into-cavity" strategy (see WO 96/27011). For this reason, another aspect of the present invention is to provide a method for producing a bispecific in a recombinant host, said method comprising the step of expressing in a recombinant host cell a nucleic acid sequence encoding at least two heavy chain polypeptides, , wherein said heavy chain polypeptides differ from each other in their constant regions sufficiently to reduce or prevent homodimer formation but increase bispecific formation.

Where the protein comprises three chains, eg, FlicAb, they can be produced by co-expression of the three chains (two heavy chains and one light chain) constituting the molecule in a single recombinant host cell.

For recombinant production of a protein herein, one or more nucleic acids encoding all chains, e.g., 2, 3, 4, etc., are isolated and placed into a replicable vector for further cloning (amplification of the DNA) or expression. is inserted Many vectors are available. Vector components generally include, but are not limited to, one or more of the following: a signal sequence, an origin of replication, one or more marker genes, an enhancer element, a promoter, and a transcription termination sequence.

In a preferred embodiment, the host cell according to the method of the present invention has a high-level expression of human immunoglobulin, ie at least 1 pg/cell/day, without the need for amplification of a nucleic acid molecule encoding a single chain in said host cell. , preferably at least 10 pg/cell/day, more preferably at least 20 pg/cell/day or more.

pharmaceutical composition

Another aspect of the present invention is to provide a pharmaceutical composition comprising one or more proteins of the present invention in admixture with a suitable pharmaceutically acceptable carrier. Pharmaceutically acceptable carriers as used herein include, but are not limited to, for example, adjuvants, solid carriers, water, buffers, or other carriers used in the art for holding therapeutic ingredients, or combinations thereof. doesn't happen

Therapeutic preparations of proteins used in accordance with the present invention may include, for example, in the form of lyophilized preparations or aqueous solutions, containing the protein of the desired degree of purity with any optional pharmaceutically acceptable carrier, excipient or stabilizer (eg, Remington's). (See Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980)). Acceptable carriers, excipients, or stabilizers are nontoxic to recipients at the dosages and concentrations employed, and include buffers such as phosphate, citrate and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride, benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol ; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptides; proteins such as serum albumin, gelatin or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose or dextrins; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose or sorbitol; salt-forming counterions such as sodium; metal complexes (eg, Zn-protein complexes); and/or nonionic surfactants such as TWEEN™, PLURONICS™ or polyethylene glycol (PEG).

Anti-CD3 antibody formulations are disclosed, for example, in US Patent Publication No. 20070065437, the entire disclosure of which is expressly incorporated herein by reference. Similar agents can be used with the proteins of the present invention. The main ingredients of these formulations are an effective pH buffer in the range of 3.0 to 6.2, salts, surfactants, and an effective amount of a bispecific with anti-CD3 specificity.

How to use

An antigen binding composition of the invention, in particular wherein said antigen binding composition is a multispecific antibody suitable for the disease being treated, eg wherein one binding moiety is specific for a tumor associated antigen for the treatment of a relevant cancer cell. bind adversarially; treating a disease, including but not limited to, infection, autoimmune disease, primary or metastatic cancer, and the like, in a regimen comprising contacting it with a binding moiety specific for a pathogen of interest, etc. for the treatment of a relevant infection; or A method is provided for reducing. Such methods include administering to a subject in need of treatment a therapeutically effective amount or effective dose of an agent of the present invention, including, but not limited to, combinations of reagents with chemotherapeutic drugs, radiotherapy or surgery.

The effective dose of the composition of the present invention for the treatment of a disease depends on the means of administration, the target site, the physiological condition of the patient, whether the patient is a human or an animal, other drugs administered, and whether the treatment is prophylactic or therapeutic. depends on many different factors, including Typically, the patient is a human, but non-human mammals such as companion animals such as dogs, cats, horses, etc., laboratory mammals such as rabbits, mice, rats, etc., will also be treated. can Therapeutic doses may be titrated to optimize safety and efficacy.

Dosage levels can be readily determined by a routinely skilled clinician, and can be altered as needed, for example, to change an individual's response to therapy. The amount of active ingredient that may be combined with the carrier materials to produce a single dosage form will vary depending upon the host treated and the particular mode of administration. Unit dosage forms generally contain between about 1 mg and about 500 mg of active ingredient.

In some embodiments, the therapeutic dose of the agent may vary in the range of about 0.0001 to 100 mg/kg of the host body weight, more typically 0.01 to 5 mg/kg of the host body weight. For example, the dose may be 1 mg/kg body weight or 10 mg/kg body weight or within the range of 1-10 mg/kg body weight. Exemplary treatment regimens entail administration once every two weeks or once a month or once every 3 to 6 months. A therapeutic entity of the present invention is typically administered in multiple doses. Intervals between single doses may be once a week, once a month, or once a year. Intervals may also be irregular, as indicated by measuring blood levels of the therapeutic entity in the patient. Alternatively, a therapeutic entity of the invention may be administered as a sustained release formulation, in which case less frequent administration is required. Dosage and frequency vary with the half-life of the polypeptide in the patient.

In prophylactic applications, relatively low doses may be administered at relatively infrequent intervals over a long period of time. Some patients continue to receive treatment for the rest of their lives. In other therapeutic applications, relatively high doses at relatively short intervals are sometimes required until progression of the disease is reduced or terminated, and preferably, until the patient shows partial or complete amelioration of symptoms of the disease. Do. Thereafter, the patient can be administered on a prophylactic regimen.

In another embodiment, the methods of the present invention treat or reduce tumor growth, tumor metastasis or tumor invasion of carcinomas, hematologic cancers such as leukemias and cancers, including lymphomas, melanomas, sarcomas, gliomas, and the like. to prevent or prevent. For prophylactic applications, the pharmaceutical composition or medicament eliminates or reduces the risk of the disease, including the biochemical, histological and/or behavioral symptoms of the disease, its complications and intermediate pathological phenotypes exhibited during the development of the disease. It is administered to a patient susceptible to, or otherwise at risk of, the disease in an amount sufficient to reduce the risk of developing the disease, reducing the severity of the disease, or delaying the onset of the disease.

Compositions for the treatment of a disease may be administered by parenteral, topical, intravenous, intratumoral, oral, subcutaneous, intraarterial, intracranial, intraperitoneal, intranasal or intramuscular means. Typical routes of administration are intravenous or intratumoral, although other routes may be equally effective.

Typically, the compositions are prepared as injectable compositions as liquid solutions or suspensions; Solid forms suitable for dissolution or suspension in a liquid vehicle prior to injection may also be prepared. The preparations may also be emulsified or encapsulated in liposomes or microparticles such as polylactide, polyglycolide or copolymers for enhanced adjuvant effect, as discussed above. Langer, Science 249: 1527, 1990 and Hanes, Advanced Drug Delivery Reviews 28: 97-119, 1997. The agents of the present invention may be administered in the form of depot injection or implantation preparations which may be formulated in a manner that permits sustained or pulsatile release of the active ingredient. Pharmaceutical compositions are formulated to be generally sterile, substantially isotonic, and fully comply with all Good Manufacturing Practices (GMP) regulations of the US Food and Drug Administration.

The toxicity of the proteins described herein can be assessed by standard pharmaceutical procedures in cell culture or laboratory animals, e.g., by determining LD ₅₀ (a dose lethal to 50% of a population) or LD ₁₀₀ (a dose lethal to 100% of a population). can be decided. The dose ratio between toxic and therapeutic effects is the therapeutic index. Data obtained from these cell culture assays and animal studies can be used to formulate a dose range that is not toxic for use in humans. Doses of the proteins described herein are preferably within a range of circulating concentrations that include effective doses with little or no toxicity. The dosage may vary within this range depending upon the dosage form employed and the route of administration utilized. The exact formulation, route of administration and dose may be selected by an individual physician in view of the patient's condition.

The pharmaceutical composition may be administered in various unit dosage forms according to the administration method. For example, unit dosage forms suitable for oral administration include, but are not limited to, powders, tablets, pills, capsules, and lozenges. It is recognized that the compositions of the present invention should be protected from digestion when administered orally. This is typically accomplished by complexing the molecules with the composition to render them resistant to acidic and enzymatic hydrolysis, or by packaging the molecules in an appropriately resistant carrier such as liposomes or a protective barrier. Means for protecting agents from digestion are well known in the art.

Compositions for administration will typically include the antibody or other ablation agent dissolved in a pharmaceutically acceptable carrier, preferably an aqueous carrier. A variety of aqueous carriers can be used, for example, buffered saline and the like. These solutions are sterile and, in general, free of undesirable substances. These compositions may be sterilized by conventional, well-known sterilization techniques. The composition may contain pharmaceutically acceptable auxiliary substances, for example, pH adjusting agents and buffers, toxicity adjusting agents, and the like, as necessary in order to approximate a physiological state, for example, sodium acetate, sodium chloride, potassium chloride, calcium chloride, sodium lactate, and the like. can be nested The concentration of active agent in these formulations can vary widely and will be selected based primarily on fluid volume, viscosity, body weight, etc., depending on the particular mode of administration chosen and the needs of the patient (see, e.g., Remington's Pharmaceutical Science (15th ed. , 1980) and Goodman & Gillman, The Pharmacological Basis of Therapeutics (Hardman et al., eds., 1996)]).

Kits comprising the active agents of the present invention, formulations thereof, and instructions for use are also within the scope of the present invention. The kit may further contain at least one additional reagent, such as a chemotherapeutic drug. Kits typically include labels indicating the intended use of the contents of the kit. The term label includes any written or recorded material supplied on or with the kit, or otherwise accompanying the kit.

The composition may be administered for therapeutic treatment. The composition is administered to the patient in an amount sufficient to substantially eliminate the targeted cells, as described above. An amount suitable to achieve this is defined as a "therapeutically effective dose", which may provide an improvement in overall survival. Single or multiple administrations of the composition may be administered as needed and according to the dosage and frequency tolerated by the patient. The specific dosage required for treatment will depend on the medical condition and medical history of the mammal as well as other factors such as age, weight, sex, route of administration, effectiveness, and the like.

Although the present invention has been described in detail, it will be apparent to those skilled in the art that various changes and modifications can be made without departing from the spirit or scope of the invention.

Example

Example 1 Genetically Engineered Mice Expressing Heavy Chain-Single Antibodies

The human IgH locus was constructed and assembled in several parts, which involved modification and ligation of the murine C region genes, which were subsequently converted to human V _H 6-DJ _H joined downstream of the realm. human V _H Two BACs with distinct clusters of genes were then co-injected with the BAC encoding the assembled (human V _H 6 -DJ _H -mouse C) fragment.

Transgenic mice carrying artificial heavy chain immunoglobulin loci in their unrearranged conformation were generated. Included constant region genes encode IgM, IgD, IgG2b, IgE, IgA and 3' enhancers. RT-PCR and serum analysis (ELISA) of transgenic mice revealed a productive rearrangement of the transgenic immunoglobulin locus and expression of heavy chain sole antibodies of various isotypes in serum. The transgenic mice were crossed with mice carrying the mutated endogenous heavy and light chain loci described above in US Patent Publication 2009/0098134 A1. Analysis of these animals demonstrated inactivation of murine immunoglobulin heavy and light chain expression and high level expression of heavy chain antibodies with variable regions encoded by human V, D, and J genes. Immunization of transgenic mice resulted in the production of high titer serologic responses of antigen-specific heavy chain antibodies. These transgenic mice expressing heavy chain antibodies with human VDJ regions were called UniRats.

Example 2 Genetically engineered mice expressing immobilized light chain antibodies

A repertoire of transgenic human antibodies was generated from H-chains with various (V _H -DJ _H ) _n rearrangements in combination with unique L-chains. To this end, the rearranged L-chain, human Vk-Jk1-Ck, was integrated in the murine germline by DNA microinjection, and the obtained transgenic animals were of the previously described murine lineage naturally expressing the human H-chain repertoire. was crossed with (Osborn et al ., 2013). This new strain of mice was named OmniFlic.

Immunization of OmniFlic mice with many different antigens produced high levels of antigen-specific IgG, similar to other transgenic mice bearing the same IgH locus. Repertoire analysis by RT-PCR identified highly variable V _H -gene rearrangements at high transcript and protein levels. In addition, only one L-chain product that is also expressed at high levels was identified.

Antigen-specific binding agents from OmniFlic were obtained by selection from NGS and cDNA libraries (yeast, E. coli , phage), which identified various H-chain transcripts upon sequencing. For expression in mammalian cells, hypermutated H-chain constructs were transfected in combination with the native transgenic Igκ sequence. No mutational changes were allowed in these rearranged Vk-Jk1-Ck, and always the same L-chain was expressed with various H-chain products to yield monoclonal human IgG.

Example 3 Generation of antigen-specific antibodies in transgenic mice;

For the generation of antigen-specific heavy chain antibodies in mice, expressing genetically engineered mice were immunized in two ways.

Immunization with recombinant extracellular domains of PD-L1 and BCMA. Recombinant extracellular domains of PD-L1 and BCMA were purchased from R&D Systems, diluted with sterile saline, and combined with adjuvant. Immunogens were combined with complete Freund's adjuvant (CFA) and incomplete Freund's adjuvant (IFA), or with Titermax and Ribi adjuvants. The first immunization (ignition) with the immunogen in CFA or Titermax was done in the left and right legs. After the first immunization with the immunogen in CFA, 2 boosters (boosters) at IFA, or 4 boosts at Ribi and 1 boost at Titermax were administered in each leg. This sequence of immunization results in the development of B cells that produce high affinity antibodies. The immunogen concentration was 10 micrograms per leg. To determine serum titers, serum was collected from mice at the final blood draw.

For the generation of anti-human CD3δε antibodies, genetically engineered mice were immunized using a DNA-based immunization protocol.

OmniFlic mice are manufactured by Aldevron, Inc. (Fargo, ND) was immunized with human and cynomolgus CD3-epsilon/delta constructs using GENOVAC Antibody Technology. Drainage lymph nodes were harvested after the last booster, and RNA was isolated. After cDNA synthesis, the IgH heavy chain antibody repertoire was characterized by next-generation sequencing and our proprietary in-house software. Candidate antigen specific VH sequences showing evidence of antigen specific positive selection were selected. Hundreds of VH sequences encoding FlicAb were selected for gene assembly and cloned into expression vectors. Subsequently, fully human FlicAb IgG1 antibody was expressed in HEK cells for analysis by flow and ELISA. Human FlicAbs were tested for binding to primary human T cells and Jurkat cells by flow. In addition, human FlicAb was tested using recombinant CD3δε protein in ELISA. All FlicAbs with positive binding to human T cells are listed in Figures 1 and 2. The selected sequences were further characterized in T cell activation assays.

Example 4 Characterization of Antibodies

The data table in Figure 3 summarizes the behavior of anti-CD3 antibodies of family 2 in monospecific and bispecific formats. Column 1 shows the sequence ID for the anti-CD3 VH sequence. Column 2 shows the MFI values for binding to Jurkat cells of the parental monospecific anti-CD3. Column 3 shows the MFI values for cyno T-cell binding of parental monospecific anti-CD3. Column 4 shows the names of the CD3:aBCMA bispecific antibodies. Column 5 shows a picogram of IL-2 released by pan T-cells stimulated by a bispecific antibody binding to BCMA protein coated on plastic at the indicated doses. Column 6 shows a picogram of IL-6 released by pan T-cells stimulated by a bispecific antibody binding to BCMA protein coated on plastic at the indicated doses. Column 7 shows a picogram of IL-10 released by pan T-cells stimulated by a bispecific antibody binding to BCMA protein coated on plastic at the indicated doses. Column 8 shows a picogram of IFN-γ released by pan T-cells stimulated by a bispecific antibody binding to BCMA protein coated on plastic at the indicated doses. Column 9 shows a picogram of TNFα released by pan T-cells stimulated by a bispecific antibody binding to BCMA protein coated on plastic at the indicated doses. Column 10 shows the EC50 of bispecific antibody-mediated U266 tumor cell lysis in the presence of human pan T-cells. Column 11 shows the percent lysis of U266 tumor cells in the presence of the bispecific antibody and human pan T-cells at a dose of 333 ng/mL of the bispecific antibody. Column 12 shows the protein binding affinity of the anti-CD3 arm of the bispecific antibody as measured by Octet. Column 13 shows the MFI values for binding to Jurkat cells of the bispecific antibody.

Example 5 Bispecific Antibody Characterization

Seven αCD3_fam2:aBCMA bispecific antibodies, each with a unique anti-CD3 arm (as indicated) and a conventional anti-BCMA arm, were tested for their ability to kill U266 BCMA+ tumor cells through redirection of activated primary T cells. . In these experiments U266 cells expressing BCMA were mixed with activated pan T-cells at a 10:1 E:T ratio with the addition of bispecific antibody. As shown in FIG. 4 , the x-axis shows the concentration of antibody used, and the y-axis shows the lysis % of tumor cells 6 hours after addition of the antibody.

A comparison of bispecific antibody-mediated tumor cell lytic activity and IL-2 cytokine release is shown in the scatter plot of FIG. 5 . The correlation between IL-2 production and U266 tumor cell lysis is R ² =0.37. A comparison of bispecific antibody-mediated tumor cell lytic activity and IFN-γ cytokine release is shown in the scatter plot of FIG. 6 . The correlation between IFN-γ production and U266 tumor cell lysis is R ² =0.53. A comparison of the bispecific antibody-mediated U266 tumor cell lytic activity and anti-CD3 binding affinity is shown in the scatter plot of FIG. 7 . The correlation between U266 killing EC50 and protein binding affinity is R ² =0.93.

Example 6 Lysis of tumor cells

The αCD3_F1F:αBCMA bispecific antibody was assayed for its ability to kill three different BCMA+ tumor cells and one BCMA-negative cell line through redirection of activated primary T cells. In these experiments, tumor cells were mixed with activated pan T-cells at a 10:1 E:T ratio with the addition of bispecific antibody. The results are shown in Figures 8A-6D. Panel A shows the killing of RPMI-8226 cells, panel B shows the killing of NCI-H929 cells, panel C shows the killing of U-266 cells, and panel D shows the killing of the negative control K562 cells. show The x-axis shows the concentration of antibody used, and the y-axis shows the percent lysis of tumor cells 6 hours after addition of the antibody.

IL-2 cytokine release levels were measured after resting human T cells were incubated with various tumor cell lines and increasing doses of αCD3_F1F:αBCMA bispecific antibody. 9A shows IL-2 release stimulated by RPMI-8226 cells, FIG. 9B shows IL-2 release stimulated by NCI-H929 cells, and FIG. 9C shows IL stimulated by U-266 cells. -2 release, and FIG. 9D shows IL-2 release stimulated by the negative control, K562 cells.

IFN-γ cytokine release levels were measured after resting human T cells were incubated with various tumor cell lines and increasing doses of the αCD3_F1F:aBCMA bispecific antibody. FIG. 10A shows IFN-γ release stimulated by RPMI-8226 cells, FIG. 10B shows IFN-γ release stimulated by NCI-H929 cells, and FIG. 10C shows IFN stimulated by U-266 cells. -γ release, and FIG. 10D shows IFN-γ release stimulated by negative control, K562 cells.

These examples are offered to provide those skilled in the art with a complete disclosure and description of how to make and use the present invention, and are not intended to limit the scope of what the inventors consider their invention to be, nor are they intended to indicate that the experiments below may be performed. It is not intended to represent all or only experiments performed. Efforts have been made to ensure accuracy with respect to numbers used (eg, amounts, temperature, etc.), but some experimental errors and deviations should be accounted for. Unless otherwise indicated, fractions are fractions by weight, molecular weight is weight average molecular weight, temperature is in degrees Celsius (°C), and pressure is at or near atmospheric.

While the invention has been described with respect to specific embodiments, it should be understood by those skilled in the art that various changes may be made and equivalents may be substituted without departing from the true spirit and scope of the invention. In addition, many modifications may be made to adapt a particular environment, material, composition of matter, process, process step or steps, to the objective, spirit and scope of the present invention. All such modifications are intended to be within the scope of the appended claims.

SEQUENCE LISTING <110> TENEOBIO, INC. <120> CD3 Binding Antibodies <130> WO/2018/052503 <140> PCT/US2017/038377 <141> 2017-06-20 <150> US 62/394,360 <151> 2016-09-14 <150> US 62 /491,908 <151> 2017-04-28 <160> 22 <170> FastSEQ for Windows Version 4.0 <210> 1 <211> 123 <212> PRT <213> Homo sapiens <400> 1 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Arg 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asp Asp Tyr 20 25 30 Ala Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Gly Ile Ser Trp Asn Ser Gly Ser Ile Gly Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Leu Tyr Tyr Cys 85 90 95 Ala Lys Asp Ser Arg Gly Tyr Gly Asp Tyr Arg Leu Gly Gly Ala Tyr 100 105 110 Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 2 <211> 123 < 212> PRT <213> Homo sapiens <400> 2 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Va l Lys Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asp Asp Tyr 20 25 30 Ala Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Gly Ile Ser Trp Asn Ser Gly Ser Ile Gly Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Leu Tyr Tyr Cys 85 90 95 Ala Lys Asp Ser Arg Gly Tyr Gly Asp Tyr Arg Leu Gly Gly Ala Tyr 100 105 110 Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 3 <211> 123 <212> PRT < 213> Homo sapiens <400> 3 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Arg 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asp Asp Tyr 20 25 30 Ala Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Gly Ile Ser Trp Asn Ser Gly Ser Ile Gly Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Lys Ser Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Leu Tyr Tyr Cys 85 90 95 Ala Lys Asp Ser Arg Gly Tyr Gly Asp Tyr Ser Arg Gly Gly Ala Tyr 100 105 110 Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 4 <211> 123 <212> PRT <213> Homo sapiens <400> 4 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Arg 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asp Asp Tyr 20 25 30 Ala Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Gly Ile Ser Trp Asn Ser Gly Ser Ile Gly Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Pro Glu Asp Thr Ala Leu Tyr Tyr Cys 85 90 95 Ala Lys Asp Ser Arg Gly Tyr Gly Asp Tyr Ser Leu Gly Gly Ala Tyr 100 105 110 Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 5 <211> 123 <212> PRT <213> Homo s apiens <400> 5 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Arg 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asp Asp Tyr 20 25 30 Ala Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Gly Ile Ser Trp Asn Ser Gly Ser Ile Gly Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Leu Tyr Tyr Cys 85 90 95 Ala Lys Asp Ser Arg Gly Tyr Gly Asp Tyr Ser Leu Gly Gly Ala Tyr 100 105 110 Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 6 <211> 123 <212> PRT <213> Homo sapiens <400> 6 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Arg 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ala Asn Tyr 20 25 30 Ala Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Gly Ile Ser Trp Asn Ser Gly Ser Ile Asp Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Leu Tyr Tyr Cys 85 90 95 Ala Lys Asp Ser Arg Gly Tyr Gly Asp Tyr Ser Arg Gly Gly Ala Tyr 100 105 110 Trp Gly Gln Gly Thr Leu Val Thr Val Ser 115 120 <210> 7 <211> 123 <212> PRT <213> Homo sapiens <400> 7 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Arg 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asn Asn Tyr 20 25 30 Ala Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Gly Ile Ser Trp Asn Ser Gly Ser Ile Gly Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Gly Glu Asp Thr Ala Leu Tyr Tyr Cys 85 90 95 Ala Lys Asp Ser Arg Gly Tyr Gly Asp Tyr Ser Arg Gly Gly Ala Tyr 100 105 110 Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ser 115 120 <210> 8 <211> 123 <212> PRT <213> Homo sapiens <400> 8 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ala Asp Tyr 20 25 30 Ala Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Gly Ile Ser Trp Asn Ser Gly Ser Ile Gly Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Leu Tyr Tyr Cys 85 90 95 Ala Lys Asp Ser Arg Gly Tyr Gly Asp Tyr Ser Arg Gly Gly Ala Tyr 100 105 110 Trp Gly Gly Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 9 <211> 123 <212> PRT <213> Homo sapiens <400> 9 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Arg 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asp Asn Tyr 20 25 30 Ala Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Gly Ile Ser Trp Asn Ser Gly Ser Ile Gly Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Lys Ser Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Leu Tyr Tyr Cys 85 90 95 Ala Lys Asp Ser Arg Gly Tyr Gly Asp Tyr Ser Arg Gly Gly Ala Tyr 100 105 110 Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 10 <211> 123 <212> PRT <213> Homo sapiens <400> 10 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Arg 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asp Asp Asn Tyr 20 25 30 Ala Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Gly Ile Ser Trp Asn Ser Gly Ser Ile Gly Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Leu Tyr Tyr Cys 85 90 95 Ala Lys Asp Ser Arg Gly Tyr Gly Asp Tyr Ser Arg Gly Gly Ala Tyr 100 105 110 Trp Gly Gln Gly Thr Leu Val Thr Val Ser 115 120 <210> 11 <211> 123 <212> PRT <213> Homo sapiens <400> 11 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Arg 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asp Asp Tyr 20 25 30 Ala Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Gly Ile Ser Trp Asn Ser Gly Ser Ile Gly Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Leu Tyr Tyr Cys 85 90 95 Ala Lys Asp Ser Arg Gly Tyr Gly Asp Tyr Ser Arg Gly Gly Ala Tyr 100 105 110 Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 12 <211> 123 <212> PRT <213> Homo sapiens <400> 12 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Arg 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asp Asp Tyr 20 25 30 Ala Met H is Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Gly Ile Ser Trp Asn Ser Gly Ser Ile Gly Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Pro Glu Asp Thr Ala Leu Tyr Tyr Cys 85 90 95 Ala Lys Asp Ser Arg Gly Tyr Gly Ser Tyr Ser Arg Gly Gly Ala Tyr 100 105 110 Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 13 <211> 123 <212> PRT <213> Homo sapiens <400> 13 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Arg 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe His Asn Tyr 20 25 30 Ala Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Gly Ile Ser Trp Asn Ser Gly Ser Ile Gly Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Leu Tyr Tyr Cys 85 90 95 Ala Lys Asp Ser Arg Gly T yr Gly Asp Tyr Ser Leu Gly Gly Ala Tyr 100 105 110 Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 14 <211> 123 <212> PRT <213> Homo sapiens <400> 14 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asp Asp Tyr 20 25 30 Ala Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Gly Ile Ser Trp Asn Ser Gly Ser Ile Gly Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Leu Tyr Tyr Cys 85 90 95 Ala Lys Asp Ser Arg Gly Tyr Gly Asp Tyr Ser Arg Gly Gly Ala Tyr 100 105 110 Cys Gly Gly Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 15 < 211> 123 <212> PRT <213> Homo sapiens <400> 15 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Arg 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asp Asn Tyr 20 25 30 Ala Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Gly Ile Ser Trp Asn Ser Gly Ser Ile Gly Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Lys Ser Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Leu Tyr Tyr Cys 85 90 95 Ala Lys Asp Ser Arg Gly Tyr Gly Asp Tyr Ser Leu Gly Gly Ala Tyr 100 105 110 Trp Gly Gln Gly Thr Leu Val Thr Val Ser 115 120 <210> 16 <211> 123 <212> PRT <213> Homo sapiens <400> 16 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Arg 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asp Asp Tyr 20 25 30 Ala Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Gly Ile Ser Trp Asn Ser Gly Ser Ile Gly Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Lys Ser Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Leu Tyr Tyr Cys 85 90 95 Ala Lys Asp Ser Arg Gly Tyr Gly Asp Tyr Arg Leu Gly Gly Ala Tyr 100 105 110 Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 17 <211> 123 <212> PRT <213> Homo sapiens <400> 17 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Arg 1 5 10 15 Ser Leu Arg Leu Ser Cys Glu Ala Ser Gly Phe Thr Phe Asp Asp Tyr 20 25 30 Ala Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Gly Ile Ser Trp Asn Ser Gly Ser Ile Gly Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Leu Tyr Tyr Cys 85 90 95 Ala Lys Asp Ser Arg Gly Tyr Gly Asp Tyr Arg Leu Gly Gly Ala Tyr 100 105 110 Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 18 <211> 123 <212> PRT <213> Homo sapiens <400> 18 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Arg 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asp Asp Tyr 20 25 30 Ala Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Gly Ile Ser Trp Asn Ser Gly Ser Ile Gly Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Pro Glu Asp Thr Ala Leu Tyr Tyr Cys 85 90 95 Ala Lys Asp Ser Arg Gly Tyr Gly Asp Tyr Arg Leu Gly Gly Ala Tyr 100 105 110 Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 19 <211> 107 <212> PRT <213> Homo sapiens <400> 19 Glu Ile Val Met Thr Gln Ser Pro Ala Thr Leu Ser Val Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Asn 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile 35 40 45 Tyr Gly Ala Ser Thr Arg Ala Thr Gly Ile Pro Ala Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Ser 65 70 75 80 Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Tyr Asn Asn Trp Pro Trp 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 100 105 <210> 20 <211> 243 <212> PRT <213> Homo sapiens <400> 20 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Val Ser Ser Tyr 20 25 30 Gly Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Pro Glu Trp Val 35 40 45 Ser Gly Ile Arg Gly Ser Asp Gly Ser Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Gln Gly Glu Asn Asp Gly Pro Phe Asp His Arg Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser Glu Val Gln Leu 115 120 125 Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu 130 135 140 Ser Cys Ala Ala Ser Gly Phe Thr Val Ser Ser Tyr Gly Met Ser Trp 145 150 155 160 Val Arg Gln Ala Pro Gly Lys Gly Pro Glu Trp Val Ser Gly Ile Arg 165 170 175 Gly Ser Asp Gly Ser Thr Tyr Tyr Ala Asp Ser Val Lys Gly Arg Phe 180 185 190 Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr Leu Gln Met Asn 195 200 205 Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Lys Gln Gly 210 215 220 Glu Asn Asp Gly Pro Phe Asp His Arg Gly Gln Gly Thr Leu Val Thr 225 230 235 240 Val Ser <210> 21 <211> 119 <212> PRT <213> Homo sapiens <400> 21 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Val Ser Ser Tyr 20 25 30 Gly Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Pro Glu Trp Val 35 4 0 45 Ser Gly Ile Arg Gly Ser Asp Gly Ser Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Gln Gly Glu Asn Asp Gly Pro Phe Asp His Arg Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 22 <211> 128 <212> PRT < 213> H. sapiens<400> 22 Gln Leu Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gln 1 5 10 15 Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Gly Ser Ile Ser Ser Gly 20 25 30 Gly His Tyr Trp Ser Trp Ile Arg Gln His Pro Gly Lys Gly Leu Glu 35 40 45 Trp Ile Gly Tyr Ile His Tyr Ser Gly Ser Thr Tyr Tyr Asn Pro Ser 50 55 60 Leu Lys Ser Arg Val Ile Ile Ser Val Asp Thr Ser Lys Asn Gln Phe 65 70 75 80 Ser Leu Lys Leu Arg Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr 85 90 95 Cys Ala Arg Trp Arg His Asp Ile Phe Ala Ala Tyr Pro Tyr Tyr Tyr 100 105 110 Tyr Gly Met Asp Val Trp Gly Gin Gly Thr Leu Val Thr Val Ser Ser 115 120 125

Claims (17)

a first binding moiety specific for human CD3δε; and
a second binding moiety specific for human BCMA;
The first binding moiety is
(i) a heavy chain variable comprising a CDR1 having the amino acid sequence of GFTFDDYA (SEQ ID NO:29), a CDR2 having the amino acid sequence of ISWNSGSI (SEQ ID NO:24), and a CDR3 having the amino acid sequence of AKDSRGYGDYRLGGAY (SEQ ID NO:41) (VH) a first polypeptide subunit comprising a domain; and
(ii) a light chain variable comprising a CDR1 having the amino acid sequence of QSVSSN (SEQ ID NO:35), a CDR2 having the amino acid sequence of GAS (SEQ ID NO:38), and a CDR3 having the amino acid sequence of QQYNNWPWT (SEQ ID NO:45) (VL) a second polypeptide subunit comprising a domain;
The second binding moiety is
(i) a VH domain comprising a CDR1 having the amino acid sequence of GFTVSSYG (SEQ ID NO: 36), a CDR2 having the amino acid sequence of IRGSDGST (SEQ ID NO: 39), and a CDR3 having the amino acid sequence of AKQGENDGPFDH (SEQ ID NO: 46) A bispecific antibody comprising a third polypeptide subunit comprising a. According to claim 1,
wherein said CDR1, CDR2 and CDR3 sequences of said VH domain of said first polypeptide subunit are present in a human VH framework. According to claim 1,
wherein said CDR1, CDR2 and CDR3 sequences of said VL domain of said second polypeptide subunit are present in a human VL framework. According to claim 1,
The CDR1, CDR2 and CDR3 sequences of the VH domain of the first polypeptide subunit are in a human VH framework, and the CDR1, CDR2 and CDR3 sequences of the VL domain of the second polypeptide subunit are human VL Bispecific antibodies present in the framework. According to claim 1,
The first polypeptide subunit is a bispecific antibody comprising a VH domain having the amino acid sequence of SEQ ID NO: 1. According to claim 1,
wherein said second polypeptide subunit comprises a VL domain having the amino acid sequence of SEQ ID NO: 19. According to claim 1,
wherein said first polypeptide subunit comprises a VH domain having the amino acid sequence of SEQ ID NO: 1 and said second polypeptide subunit comprises a VL domain having the amino acid sequence of SEQ ID NO: 19. According to claim 1,
wherein said third polypeptide subunit comprises a VH domain having the amino acid sequence of SEQ ID NO: 21. According to claim 1,
wherein the third polypeptide subunit is a bispecific antibody having the amino acid sequence of SEQ ID NO: 20. 6. The method of claim 5,
wherein said third polypeptide subunit comprises a VH domain having the amino acid sequence of SEQ ID NO: 21. 6. The method of claim 5,
wherein the third polypeptide subunit is a bispecific antibody having the amino acid sequence of SEQ ID NO: 20. 7. The method of claim 6,
wherein said third polypeptide subunit comprises a VH domain having the amino acid sequence of SEQ ID NO: 21. 7. The method of claim 6,
wherein the third polypeptide subunit is a bispecific antibody having the amino acid sequence of SEQ ID NO: 20. a first binding moiety specific for human CD3δε; and
a second binding moiety specific for human BCMA;
The first binding moiety comprises: a first polypeptide subunit comprising a VH domain having the amino acid sequence of SEQ ID NO:1; and a second polypeptide subunit comprising a VL domain having the amino acid sequence of SEQ ID NO:19;
wherein said second binding moiety comprises a third polypeptide subunit comprising a VH domain having the amino acid sequence of SEQ ID NO:21. 15. The method of claim 14,
wherein the third polypeptide subunit comprises a first VH domain having the amino acid sequence of SEQ ID NO:21 and a second VH domain having the amino acid sequence of SEQ ID NO:21, wherein the first VH domain and the second VH domain A bispecific antibody arranged in a tandem structure. 16. The method of claim 15,
wherein the first VH domain and the second VH domain of the third polypeptide subunit are arranged in a tandem structure and connected by a linker. a first binding moiety specific for human CD3δε; and
a second binding moiety specific for human BCMA;
The first binding moiety comprises: a first polypeptide subunit comprising a VH domain having the amino acid sequence of SEQ ID NO:1; and a second polypeptide subunit comprising a VL domain having the amino acid sequence of SEQ ID NO:19;
wherein said second binding moiety comprises a third polypeptide subunit having the amino acid sequence of SEQ ID NO:20.

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